DNA encoding PACAP receptor protein and method for preparing said protein

ABSTRACT

This invention discloses a PACAP receptor protein or a salt thereof, a DNA comprising a DNA fragment coding for the protein, a method for preparing the protein, antibody against the protein and use of the protein, DNA and antibodies. A PACAP receptor protein was purified from the bovine cerebrum. DNAs coding for PACAP receptor proteins were isolated from bovine, rat and human cDNA libraries, and their nucleotide sequences were determined. The PACAP receptor proteins and the DNAs coding for the proteins of the present invention can be used for (1) acquisition of antibodies and antisera, (2) construction of expression systems of recombinant receptor proteins, (3) development of receptor binding assay systems using said expression systems and screening of potential compounds for drugs, (4) execution of drug design based on the comparison of ligands and receptors which are structurally similar to each other, (5) preparation of probes and PCR primers in gene diagnosis, and the like.

This is a divisional of application(s) Ser. No. 08/202,986 filed on Feb.25, 1994 now abandoned.

FIELD OF THE INVENTION

The present invention relates to a pituitary adenylate cyclaseactivating polypeptide (hereinafter referred to "PACAP") receptorprotein (hereinafter may be referred to "PACAP receptor protein") or asalt thereof which is capable of binding a PACAP, a DNA comprising a DNAfragment coding for said protein, a method for preparing said protin orthe salt thereof, and use of said protein and said DNA.

BACKGROUND OF THE INVENTION

PACAP was first isolated from the hypothalami of sheep as a peptidepromoting adenylate cyclase activity of the pituitary glands BiochemicalBiophysical Research Communications, 164, 567-574 (1989)!. PACAP firstisolated was one consisting of 38 amino acid residues. However, thepresence of PACAP consisting of 27 residues on the N-terminal side wasalso revealed. Both are nearly equal in adenylate cyclase activatingability to each other. The former is referred to as PACAP38, and thelatter is referred to as PACAP27. The expression of a PACAP having thesame structure as that of sheep was also proved in humans, whichsuggested that PACAPs are important peptides conserved beyond speciesBiochem. Biophys. Res. Commun., 166, 81-89 (1990)!. For the distributionthereof in organs, they are observed not only in the brain hypothalami,but also in the pituitary glands, the testes and the adrenals(Endocrinology, 129, 2787-2789). At present, PACAPs such as PACAP27 toPACAP38 (U.S. Pat. No. 5,128,242) and PACAP23 to PACAP26 (EuropeanPatent Publication No. 0467279A3) have been reported.

Physiological actions of PACAPs diversely varies according to theiroccurrence sites. Various actions of the PACAPs as described below havehitherto been reported:

(1) Promotion of cAMP production in primary culture cells of the ratpituitary glands A. Miyata et al., Biochem. Biophys. Res. Commun., 164,567-574 (1989)!;

(2) Promotion of secretion of GH, ACTH, PRL and LH in the rat pituitarygland superfusion process A. Miyata et al., Biochem. Biophys. Res.Commun., 164, 567-574 (1989)!;

(3) Production of cAMP in adrenomedullary chromaffinoma-derived cellsPC12h and promotion of neurite outgrowth T. Watanabe et al., Biochem.Biophys. Res. Commun., 173, 252-258 (1990), and K. Okazaki et al., FEBSLetters, 298, 49-56 (1992)!;

(4) Promotion of interleukin-6 production in pituitary gland culturecells I. Tatsuno et al., Endocrinology, 129, 1797-1804 (1991)!; and

(5) Promotion of cAMP production in primary culture of rat astrocytesand promotion of action preventing nerve cell death Biochem. Biophys.Res. Commun., 168, 1027-1033 (1990)!.

In order for PACAP to exhibit its action, the presence of a receptorspecific for PACAP in target organs and cells is indispensable.

Receptor binding experiments using radioactive iodine-labeled PACAP27((¹²⁵ I! PACAP27) have proved the presence of a PACAP receptor. Namely,when a membrane fraction prepared from a tissue is mixed with ¹²⁵ I!PACAP27 and reacted for an appropriate period of time, binding of ¹²⁵ I!PACAP27 to the membrane fraction is observed. This binding is inhibitedby unlabeled PACAP27 or PACAP38, but not inhibited by VIP, an analogouspeptide of the PACAPs. This result suggests that a substancespecifically binding to the PACAPs occurs in the tissue. Such bindingactivity is highest in membrane fractions of the brain hypothalamic andalso observed in the pituitary glands, the adrenals and the likeEndocrinology, 127, 272-277 (1990)!. Further, a body of PACAP bindingactivity observed in membrana cerebri fractions, namely a receptor, isdeduced to be a protein having a molecular weight of 57,000 from atechnique (so-called affinity-label experiment) comprising binding ¹²⁵I! PACAP27 to the membrana cerebri fraction, crosslinking ¹²⁵ I! PACAP27and the body of its binding activity with a crosslinking reagent, thensubjecting the product to polyacrylamide gel electrophoresis in thepresence of sodium dodecylsulfate, and analyzing by autoradiographyBiochem. Biophys. Res. Commun., 171, 838-844 (1990)!.

It is expected that clarification of some fundamental properties of thisspecific receptor allows elucidation of additional various properties ofthe PACAP receptor to proceed more than before. In particular, cloningof cDNA coding for the receptor protein and structure analysis thereofenable elucidation of the mechanism of its mutual interaction with aligand, production of receptor-agonists and antagonists and detailedanalysis of sites of action by in situ hybridization using said cDNA.Although cloning of the VIP, secretin and growth hormone releasingfactor receptor proteins cDNA has been reported, the cloning of thePACAP receptor has not. These three kinds of bioactive peptides havealso showed a capital similarity in the structures of their receptorproteins. For the PACAP receptors, however, cloning of cDNA has hithertonot been carried out.

Recently, the following five documents reported amino acid sequences fora rat PACAP receptor protein and nucleotide sequences of DNAs coding forthe protein Document 1: Biochemical and Biophysical ResearchCommunications, 194, 1, pp.133-143, 1993; Document 2: Federation ofEuropean Biochemical Societies (FEBS), 329, 1 and 2, pp. 99-105;Document 3: Proceedings of the National Academy of Science, U.S.A., 90,pp. 6345-6349, 1993; Document 4: Nature, 365, pp. 170-175, 1993 andDocument 5: Neuron, 11, pp.333-342, 1993). Among them, the amino acidsequences and the nucleotide sequences described in the Documents 1, 2,4 and 5 are identified with the amino acid sequence for a rat PACAPreceptor protein and with the nucleotide sequence for a DNA coding forthe protein. The amino acid sequence described in Document 3 isdifferent from the amino acid sequence of the present invention for arat PACAP receptor protein in one amino acid, and the nucleotidesequence of Document 3 is also different from the nucleotide sequence ofthe present invention in one nucleotide. All of the five documents werepublished after Jun. 24, 1993 which is one of the priority dates of thepresent invention.

In general, when specific binding substances such as receptors arepurified, affinity column chromatography applying its mutual interactionwith the specific binding substance (for example, ligands for receptors)are frequently used. A process using an affinity column in which aligand is fixed on a carrier is simplest. However, many successfulexamples of complicated affinity chromatography are known in which thespecific mutual interaction between avidin and biotin is utilized forpurification of receptors. This process comprises synthesizing abiotinylated ligand in which biotin is bound to an appropriate site, andspecifically capturing a receptor on a carrier on which avidin is fixedthrough the biotinylated ligand Methods in Enzymology, 184, 244-274(1990)!. This process suffers from the problem of designing thebiotinylated ligand having affinity for both the receptor and avidin,and examination is required in purifying PACAP receptor.

PACAP38 and PACAP27 are peptides represented by the following amino acidsequences, respectively: ##STR1##

SUMMARY OF THE INVENTION

In order to further elucidate the properties of the PACAP receptorprotein which is a giant protein molecule and to collect usefulinformation for development of drugs, purification of said protein,structure analysis by cDNA cloning and construction of its expressionsystem are indispensable. As described above, the presence of a proteinshowing high affinity for the PACAP, namely the PACAP receptor protein,has been known in the animal tissues. However, no report has so far beenmade that the PACAP receptor protein has yet to be obtained.

An object of the present invention is to purify the PACAP receptorprotein and to clone a DNA coding for the PACAP receptor protein. Ifdetailed information about the facts of the structure and functions ofsaid protein is obtained, not only development of diagnostic methods forneuropathy such as Alzheimer's disease induced by a decrease in PACAPconcentration is enabled by detecting the PACAP concentration in vivo,but also compounds activating PACAP receptor other than the known PACAPproteins or compounds antagonizing binding of a PACAP to a PACAPreceptor can be enabled by using the PACAP receptor protein and the DNAcording for said protein. In addition, gene therapeutic composition forneuropathy such as Alzheimer's disease can be enabled by using said DNA.

The present inventors conducted intensive investigations, in view of theabove-mentioned situation. As a result, bovine PACAP receptor proteinwas prepared unexpectedly efficiently by affinity chromatography usingbiotinylated PACAPs (particularly, biotinylated PACAP27). Further,synthetic DNA was prepared as a probe, based on the N-terminal aminoacid sequence of the purified bovine PACAP receptor protein, and abovine brain cDNA library was screened to clone cDNA of bovine PACAPreceptor. As a result, the present inventors first succeeded in cloninga bovine cDNA encoding the receptor protein for PACAP from the bovinebrain cDNA library and in determining a nucleotide sequence of atranslation region thereof. Further, the present inventors elucidatedthe amino acid sequence of bovine PACAP receptor protein from this cDNA,and succeeded in pioneering the mass production thereof by recombinanttechnology.

Furthermore, the present inventors based on the similarity of thestructure of PACAPs to that of VIP, secretin and growth hormonereleasing factor, and deduced that receptors for the PACAPs would alsoshow a similar structure to these, from the fact that the receptorsalready elucidated extremely resemble in structure among VIP, secretinand growth hormone releasing factor. Then, using as a probe cDNA of theVIP receptor having a higher similarity in structure as a ligand, cDNAof PACAP receptors was screened by homology screening. As a result, thepresent inventors first succeeded in cloning cDNA coding for rat PACAPreceptor protein from a rat brain cDNA library, and in determining anucleotide sequence of a translation region thereof. Further, thepresent inventors elucidated the amino acid sequence of rat PACAPreceptor protein from this cDNA, and succeeded in pioneering the massproduction thereof by recombinant technology.

In addition, the present inventors succeeded in cloning cDNA coding forhuman PACAP receptor protein from a human pituitary cDNA library, usingas a probe synthetic DNA prepared based on the amino acid sequence(sequence consisting of 16 amino acids) on the N-terminal side of thepurified bovine PACAP receptor protein, and in determining a nucleotidesequence of a translation region thereof. Then, the present inventorselucidated the amino acid sequence of human PACAP receptor protein fromthis cDNA, produced this in large amounts by recombinant technology, andsucceeded in pioneering the screening of compounds activating PACAPreceptors or compounds antagonizing PACAP receptors by use of humanPACAP receptor protein thus produced.

Namely, the present invention provides:

(1) A receptor protein capable of binding a PACAP or a salt thereof;

(2) The receptor protein of (1), wherein the receptor is endogenous torat, bovine or human;

(3) The receptor protein of (1) which comprises an amino acid sequencecontaining at least one member selected from the group consisting of theamino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 or a salt thereof;

(4) The receptor protein of (1) which comprises an amino acid sequencecontaining the amino acid sequence of SEQ ID NO: 13 or a salt thereof;

(5) The receptor protein of (1) which comprises an amino acid sequencehaving 90 to 100% homology as determined by sequence analysis with atleast one member selected from the group consisting of the amino acidsequences of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20,SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 28 or a saltthereof;

(6) The receptor protein of (1) which comprises an amino acid sequenceselected from the group consisting of the amino acid sequences of SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:28 and SEQ ID NO: 29 or a salt thereof;

(7) A receptor fragment containing a sufficient portion of the receptorof (1) to bind PACAP or a salt thereof;

(8) The receptor fragment of (7) selected from the group consisting of

(i) peptides having the amino acid sequence consisting of the 38th to164th, 223rd to 232nd, 303rd to 317th or 416th to 424th amino acidresidues of SEQ ID NO: 15,

(ii) peptides having the amino acid sequence consisting of the 38th to164th, 223rd to 232nd, 303rd to 317th or 388th to 397th amino acidresidues of SEQ ID NO: 17,

(iii) peptides having the amino acid sequence consisting of the 20th to146th, 205th to 214th, 286th to 299th or 369th to 378th amino acidresidues of SEQ ID NO: 19,

(iv) peptides having the amino acid sequence consisting of the 20th to146th, 205th to 214th, 286th to 299th or 397th to 406th amino acidresidues of SEQ ID NO: 21, and

(v) peptides having the amino acid sequence consisting of the 78th to204th, 263rd to 272nd, 342nd to 357th or 427th to 436th amino acidresidues of SEQ ID NO: 23; or a salt thereof;

(9) An isolated DNA coding for a receptor protein capable of binding aPACAP;

(10) The DNA of (9) wherein the receptor protein comprises the aminoacid sequence of SEQ ID NO: 14, SEQ ID NO 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO 18, SEQ ID NO: 19, SEQ ID NO 20, SEQ ID NO: 21, SEQ ID NO22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO 25, SEQ ID NO: 26, SEQ ID NO27, SEQ ID NO: 28 or SEQ ID NO 29;

(11) The DNA of (9) comprising the nucleotide sequence of SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO 32,

(13) A vector containing the DNA of (9);

(14) A transformant containing the DNA of (9);

(15) A method for preparing the receptor protein or the salt thereof of(1) comprising cultivating a transformant containing a DNA encoding saidprotein under conditions suitable for expression of said protein andrecovering said protein;

(16) A method for purifying the receptor protein or the salt thereof of(1) comprising subjecting a sample containing unpurified receptorprotein to affinity chromatography using a biotinylated PACAP;

(17) The method of (16) comprising the steps of:

(a) preparing a membrane protein fraction from an animal tissue or cell,

(b) solubilizing the membrane protein fraction obtained in step (a),

(c) subjecting the solubilized membrane protein fraction obtained instep (b) to anion exchange chromatography and/or hydroxyapatitechromatography, and

(d) subjecting the active fraction obtained in step (c) to affinitychromatography using a biotinylated PACAP;

(18) The method of (17), in which the animal tissue is a bovinecerebrum;

(19) A method for preparing the receptor protein or the salt thereof of(1) comprising condensing a partial peptide fragment or a single aminoacid corresponding to a portion of the protein as claimed in claim 1with a residual moiety, and removing a protective group as so desiredwhen the product has the protective group, until said protein isobtained;

(20) A diagnostic composition for neuropathy comprising the PACAPreceptor protein or the salt thereof of (1), or the receptor fragment orthe salt thereof of (7);

(21) The diagnostic composition of (20) which is a diagnosticcomposition for Alzheimer's disease;

(22) A gene therapeutic composition comprising the DNA of (9);

(23) The gene therapeutic composition of (22) to be administered to apatient whose an amount of PACAP receptor protein is decreased, toincrease the amount of PACAP receptor protein;

(24) A method of diagnosis for neuropathy comprising contacting a sampleto be tested with a receptor protein capable of binding a PACAP proteinand measuring the amount of PACAP binding to the receptor protein;

(25) The method of diagnosis of (24), wherein the receptor protein is areceptor fragment of (7);

(26) The method of (24) wherein a decrease in PACAP concentration is anindication of the presence of Alzheimer's disease;

(27) A method of using the DNA of (9) to transform a cell;

(28) The method of (27) wherein the cell is transformed in vitro;

(29) The method of (27) wherein the cell is transformed in vivo;

(30) The method of (27), in which the expression of the DNA increasesthe amount of PACAP receptor protein;

(31) A method for determining

(i) an effect of a test compound on PACAP receptor activity comprisingcomparing PACAP receptor activities in cases of (a) and (b);

(a) contacting PACAP receptor with a PACAP;

(b) contacting PACAP receptor with a PACAP and a test compound, or

(ii) an effect of a test compound on binding of PACAP to PACAP receptorcomprising comparing an amount of binding of PACAP to PACAP receptor incases of (a) and (b);

(a) contacting PACAP receptor with a PACAP;

(b) contacting PACAP receptor with a PACAP and a test compound;

(32) The method of (31) wherein the PACAP receptor is a protein of (1);

(33) The method of (31) wherein the PACAP receptor is a receptorfragment of (7);

(34) The method of (31) wherein the PACAP receptor is a protein producedby cultivating a transformant containing the DNA of (9);

(35) The method of (31) which is a method for screening a compoundactivating PACAP receptor or a compound antagonizing binding of a PACAPto a PACAP receptor;

(36) An assay for quantifying a test compound's effect

(i) on PACAP receptor activity comprising comparing an amount of PACAPreceptor activation in cases of (a) and (b);

(a) contacting PACAP receptor with a PACAP;

(b) contacting PACAP receptor with a PACAP and a test compound, or

(ii) on binding of PACAP to PACAP receptor comprising comparing anamount of binding of PACAP to PACAP receptor in cases of (a) and (b);

(a) contacting PACAP receptor with a PACAP;

(b) contacting PACAP receptor with a PACAP and a test compound;

(37) A compound or a salt thereof obtained by the method of (31);

(38) The compound or a salt thereof of (37) which is a compoundactivating PACAP receptor or a compound antagonizing binding of a PACAPto a PACAP receptor;

(39) A pharmaceutical composition for neuropathy comprising an effectiveamount of the compound or the salt thereof of (37);

(40) The pharmaceutical composition of (39), wherein the neuropathy isAlzheimer's disease;

(41) An antibody to a receptor protein capable of binding a PACAP, apartial peptide thereof or a salt thereof;

(42) The antibody of (41) which is a monoclonal antibody selected fromthe group consisting of PRN1-25a, PRN1-109a and PRN1-159a;

(43) Hybridoma which produces a monoclonal antibody of (42);

(44) A signal peptide selected from the group of peptides consisting ofa peptide which has 1st to 37th amino acid sequence of SEQ ID NO:15, apeptide which has 1st to 37th amino acid sequence of SEQ ID NO:17, apeptide which has 1st to 19th amino acid sequence of SEQ ID NO:19, apeptide which has 1st to 19th amino acid sequence of SEQ ID NO:21, apeptide which has 1st to 77th amino acid sequence of SEQ ID NO:23, apeptide which has 1st to 77th amino acid sequence of SEQ ID NO:25, apeptide which has 1st to 77th amino acid sequence of SEQ ID NO:27, apeptide which has 1st to 77th amino acid sequence of SEQ ID NO:29, apeptide which has 58th to 77th amino acid sequence of SEQ ID NO:23, apeptide which has 58th to 77th amino acid sequence of SEQ ID NO:25, apeptide which has 58th to 77th amino acid sequence of SEQ ID NO:27 and apeptide which has 58th to 77th amino acid sequence of SEQ ID NO:29; or asalt thereof;

(45) A DNA which codes for a peptide of (44);

(46) A DNA of (45) which is selected from the group consisting of a DNAwhich has 1st to 111th nucleotide sequence of SEQ ID NO:30, a DNA whichhas 1st to 111th nucleotide sequence of SEQ ID NO:31, a DNA which has1st to 57th nucleotide sequence of SEQ ID NO:32, a DNA which has 1st to57th nucleotide sequence of SEQ ID NO:33, a DNA which has 1st to 231stnucleotide sequence of SEQ ID NO:34, a DNA which has 1st to 231stnucleotide sequence of SEQ ID NO:35, a DNA which has 1st to 231stnucleotide sequence of SEQ ID NO:36, a DNA which has 1st to 231stnucleotide sequence of SEQ ID NO:37, a DNA which has 172nd to 231stnucleotide sequence of SEQ ID NO:34, a DNA which has 172nd to 231stnucleotide sequence of SEQ ID NO:35, a DNA which has 172nd to 231stnucleotide sequence of SEQ ID NO:36 and a DNA which has 172nd to 231stnucleotide sequence of SEQ ID NO:37;

(47) A biotinylated PACAP;

(48) The biotinylated PACAP of (47) which is represented by thefollowing formula: ##STR2## (49) The biotinylated PACAP of (47) or (48),in which the PACAP is PACAP27; and

(50) A method for preparing the biotinylated PACAP of (47) comprisingreacting a PACAP derivative in which a cysteine residue is introducedinto the carboxyl terminus of a PACAP with a biotinylating reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a restriction enzyme cleavage map of a bovine PACAPreceptor cDNA clone, wherein A indicates AvaII, Ac indicates AccI, Bindicates BamHI, Ba indicates BalI, and S indicates SmaI;

FIG. 2 shows a nucleotide sequence (SEQ ID NO:38) of a bovine cDNAclone, pBPR-T, encoding receptor protein for PACAP and the predictedamino acid sequence (SEQ ID NO:15). A signal sequence is deduced to becleaved at that position indicated by the upward arrow ▴. Disappearanceof the region between the open triangles Δ is observed for pBPR-TD;

FIG. 3 shows a nucleotide sequence (SEQ ID NO:39) of a bovine cDNAclone, pBPR-T, encoding receptor protein for PACAP and the predictedamino acid sequence (SEQ ID NO:17). A signal sequence is deduced to becleaved at the position indicated by the upward arrow ▴. Insertion wasobserved at the position indicated by the open triangle Δ for pBPR-TD;

FIG. 4 shows the N-terminal amino acid sequence of bovine PACAP receptorprotein and an amino acid sequence deduced from pBPR-T and pBPR-TD forcomparison;

FIG. 5 shows graphs in which the degree of hydrophobicity of bovinePACAP receptor protein, encoded by pBPR-T and pBPR-TD, is shown as anindex. A and B corresponds to pBPR-T and pBPR-TD, respectively. Thenumerals 1 to 7 indicate transmembrane domains presumed from the degreeof hydrophobicity. The upward arrow ▴ indicates the position of asequence which does not exist in pBPR-TD, but exists in pBPR-T;

FIG. 6 shows a restriction enzyme cleavage map of a rat PACAP receptorcDNA clone, wherein N indicates NcoI, P indicates PstI, and B indicatesBamHI;

FIG. 7 shows a nucleotide sequence (SEQ ID NO:40) of rat PACAP receptorcDNA contained in pRPACAPR46-5 and an amino acid sequence (SEQ ID NO:19)of a translation frame derived therefrom. A signal sequence is deducedto be cleaved at the position indicated by the upward arrow ▴. Insertionis observed at the position indicated by the open triangle Δ forpRPACAPR12;

FIG. 8 is a continuation of FIG. 7;

FIG. 9 shows a nucleotide sequence (SEQ ID NO:41) of rat PACAP receptorcDNA contained in pRPACAPR12 and an amino acid sequence (SEQ ID NO:21)of a translation frame derived therefrom. A signal sequence is deducedto be cleaved at the position indicated by the upward arrow A. Thesequence between the open triangles Δ is a sequence not existing inpRPACAPR46-5 and characteristic of pRPACAPR12;

FIG. 10 is a continuation of FIG. 9;

FIG. 11 shows the N-terminal amino acid sequence of bovine PACAPreceptor protein and the N-terminal amino acid sequence of rat PACAPreceptor protein for comparison;

FIG. 12 shows a restriction enzyme cleavage map of a human PACAPreceptor cDNA clone, wherein N indicates NcoI, ScI indicates SacI, Bgindicates BglII, Hp indicates HpaI, ScII indicates SacII, ET22 indicatesEcoT22I, and Bs indicates BspEI;

FIG. 13 shows a nucleotide sequence (SEQ ID NO:42) of human PACAPreceptor Type I-A cDNA coded with pTS847-1 and an amino acid sequence(SEQ ID NO:23) of a translation frame derived therefrom;

FIG. 14 shows the N-terminal amino acid sequence of bovine PACAPreceptor protein and the N-terminal amino acid sequence deduced fromhuman PACAP receptor protein cDNA for comparison;

FIG. 15 shows nucleotide sequences of pHPR15A, pHPR55A and pHPR66Pencoding a portion of human PACAP receptor Type I-B (nucleotide 400-441of SEQ ID NO:25), Type I-B2 (nucleotide 400-440 of SEQ ID NO:27) andType I-C (nucleotide 400-441 of SEQ ID NO:29) respectively and predictedamino acid sequences of a translation frame. The region between twoarrows shows an insertion sequence into human PACAP receptor Type I-ARat Types I-B (342-383 of SEQ ID NO:2);

FIG. 16 shows a nucleotide sequence (SEQ ID NO:45) of cDNA of humanPACAP receptor Type I-B and a predicted amino acid sequence (SEQ IDNO:29) of a translation frame. An underlined region is a sequenceinserted by an alternative splicing;

FIG. 17 shows a nucleotide sequence (SEQ ID NO:44) of cDNA of humanPACAP receptor Type I-B2 and a predicted amino acid sequence (SEQ IDNO:27) of a translation frame. An underlined region is a sequenceinserted by an alternative splicing;

FIG. 18 shows a nucleotide sequence (SEQ ID NO:43) of cDNA of humanPACAP receptor Type I-C and a predicted amino acid sequence (SEQ IDNO:29) of a translation frame. An underlined region is a sequenceinserted by an alternative splicing;

FIG. 19 shows graphs in which the degree of hydrophobicity of rat PACAPreceptor protein encoded by pRPACAPR46-5 and pRPACAPR12 is shown as anindex. A and B correspond to pRPACAPR46-5 and pRPACAPR12, respectively.The numerals 1 to 7 indicate portions deduced to be domains passingthrough a cell membrane from the degree of hydrophobicity. The upwardarrow ▴ indicates the position of a sequence which does not exist inpRPACAPR46-5, but exists in pRPACAPR12;

FIG. 20 shows an amino acid sequence (amino acids 10-467 of SEQ IDNO:19) of rat PACAP receptor protein encoded by pRPACAPR46-5, and anamino acid sequence (SEQ ID NO:56) of rat VIP receptor protein forcomparison. A group of amino acids 1 to 5 shown in the upper portion ofthe figure are regarded as equivalent to one another. Residues in whichagreement is observed, including these amino acids, are given asterisks(*). The upper lines indicate the amino acid sequence of the PACAPreceptor protein encoded by pRPACAPR46-5, and the lower lines indicatethe sequence of rat VIP receptor. The numerals given above and under therespective sequences indicate the positions from the N-termini;

FIG. 21 shows a graph in which the degree of hydrophobicity of humanPACAP receptor protein coded with pTS847-1 is shown as an index. Thenumerals 1 to 7 indicate portions deduced to be domains passing througha cell membrane from the degree of hydrophobicity;

FIG. 22 shows amino acid sequences of human PACAP receptor protein(amino acids 1-525 of SEQ ID NO:23), bovine PACAP receptor protein(amino acids 1-513 of SEQ ID NO:15) and rat PACAP receptor protein(amino acids 1-495 of SEQ ID NO:21) for comparison. The arrow indicatesa cleavage site of a signal peptide;

FIG. 23 is a series of graphs in which the degree of hydrophobicity ofbovine PACAP receptor protein encoded by pBPR-T is shown as an index;

FIG. 24 is a series of graphs in which the degree of hydrophobicity ofbovine PACAP receptor protein encoded-by pBPR-TD is shown as an index;

FIG. 25 is a series of graphs in which the degree of hydrophobicity ofrat PACAP receptor protein encoded by pRPACAPR46-5 is shown as an index;

FIG. 26 is a series of graphs in which the degree of hydrophobicity ofrat PACAP receptor protein encoded by pRPACAPR12 is shown as an index;

FIG. 27 is a series of graphs in which the degree of hydrophobicity ofhuman PACAP receptor protein encoded by pTS847-1 is shown as an index;

FIG. 28 is a graph showing absorption curves of biotinated PACAP27 byHPLC. Peak 1 of (A) indicates a peak of PACAP27-Cys, peak 3 of (B)indicates a peak of biotinated PACAP27-Cys, and peak 2 of (C) indicatesa peak of the biotinating reagent;

FIG. 29 is a graph showing results of the antagonistic bindingexperiments of PACAP27 (◯) and biotinated PACAP27 (). The numerals onthe abscissa indicate the concentrations (log M) of PACAP27 andbiotinated PACAP27, and the numerals on the ordinate indicate thebinding (%) of ¹²⁵ I!-PACAP27, taking the specific binding as 100, wheneach peptide is added so as to give the final concentrations on theabscissa;

FIG. 30 is a graph showing Scatchard plot analysis of results of thesaturation binding experiments of purified bovine PACAP receptor proteinand membrane binding bovine receptor protein using ¹²⁵ I!-PACAP27. Kdindicates the dissociation constant;

FIG. 31 is a graph showing results of the antagonistic bindingexperiment of purified bovine PACAP receptor protein. The numerals onthe abscissa indicate the concentrations (log M) of PACAP38, PACAP27 andVIP, and the numerals on the ordinate indicate the binding (%) of ¹²⁵I!-PACAP27, taking the specific binding as 100, when each peptide isadded so as to give the final concentrations on the abscissa;

FIG. 32 shows analysis results of purified bovine PACAP receptor proteinby polyacrylamide electrophoresis in the presence of sodiumdodecylsulfate;

FIG. 33 is a graph showing results of the saturation binding experimentin a membrane fraction of CHO cells transfected with the bovine PACAPreceptor protein cDNA(pBPR-T). The numerals on the abscissa indicate theconcentration of ¹²⁵ I!-PACAP27 added, and the numerals on the ordinateindicate the concentration of ¹²⁵ I!-PACAP27 specifically bound to themembrane fraction;

FIG. 34 is a graph showing Scatchard plot in the membrane fraction ofCHO cells transfected with the bovine PACAP receptor protein cDNA(pBPR-T);

FIG. 35 is a graph showing results of the competitive bindingexperiments of PACAP27, PACAP38 and VIP to ¹²⁵ I!-PACAP27 in themembrane fraction of CHO cells transfected with the bovine PACAPreceptor protein cDNA (pBPR-T). The numerals on the abscissa indicatethe concentrations (log M) of PACAP27, PACAP38 and VIP, and the numeralson the ordinate indicate the binding (%) of ¹²⁵ I!-PACAP27, taking thespecific binding as 100, when each peptide is added so as to give thefinal concentrations on the abscissa;

FIG. 36 is a graph showing changes in the amounts of intracellularcyclic AMP of CHO cells transfected with the bovine PACAP receptorprotein cDNA (pBPR-T) produced by PACAP27, PACAP38 and VIP. The numeralson the abscissa indicate the concentrations (log M) of PACAP27, PACAP38and VIP, and the numerals on the ordinate indicate the relativeconcentrations of cyclic AMP in the transformant CHO cells treated withpeptides having respective concentrations, taking the concentration ofcyclic AMP in untreated transformant CHO cells as 1;

FIG. 37 is a graph showing changes in the amounts of intracellularinositol phosphate of CHO cells transfected with the bovine PACAPreceptor protein cDNA (pBPR-T) produced by PACAP27, PACAP38 and VIP. Thenumerals on the abscissa indicate the concentrations (log M) of PACAP27,PACAP38 and VIP, and the numerals on the ordinate indicate the relativeconcentrations of inositol phosphate in transformant CHO cells treatedwith peptides having respective concentrations, taking the concentrationof inositol phosphate in untreated transformant CHO cells as 1;

FIG. 38 shows results of northern hybridization using RNA prepared fromthe rat brains, lungs, livers, kidneys and testes, and a rat PACAPreceptor protein cDNA probe. The bands represent that the RNA preparedfrom the rat brains, lungs, livers, kidneys and testes, and the ratPACAP receptor protein cDNA probe exhibit cross reaction. The numeralson the left indicate the size of a molecular weight marker;

FIG. 39 shows results of measurements of the radioactivity, wherein eachcolumn indicates the binding of each CHO cell product with ¹²⁵I!-PACAP27 when cultured in each of the following combinations:

Column 1: untreated CHO cells+ ¹²⁵ I!-PACAP27

Column 2: untreated CHO cells+ ¹²⁵ I!-PACAP27+cold PACAP27

Column 3: untreated CHO cells+ ¹²⁵ I!-PACAP27+cold VIP

Column 4: pRPR3-A-introduced CHO cells+ ¹²⁵ I!-PACAP27

Column 5: pRPR3-A-introduced CHO cells+ ¹²⁵ I!-PACAP27+cold PACAP27

Column 6: pRPR3-A-introduced CHO cells+ ¹²⁵ I!-PACAP₂₇ +cold VIP

Column 7: pRPR4-B-introduced CHO cells+ ¹²⁵ I!-PACAP27

Column 8: pRPR4-B-introduced CHO cells+ ¹²⁵ I!-PACAP27+cold PACAP27

Column 9: pRPR4-B-introduced CHO cells+ ¹²⁵ I!-PACAP27+cold VIP

Column 10: rat VIP receptor cDNA-introduced CHO cells+ ¹²⁵ I!-PACAP27

Column 11: rat VIP receptor cDNA-introduced CHO cells+ ¹²⁵I!-PACAP27+cold PACAP27

Column 12: rat VIP receptor cDNA-introduced CHO cells+ ¹²⁵I!-PACAP27+cold VIP;

FIG. 40 are graphs showing results of competitive binding experiments.(A) is a graph showing results of the competitive binding experiments ofPACAP27 and VIP to ¹²⁵ I!-PACAP27 in a membrane fraction of CHO cellstransfected with rat PACAP receptor protein cDNA (pRPR3-A). (B) is agraph showing results of the competitive binding experiments of PACAP27and VIP to ¹²⁵ I!-PACAP27 in a membrane fraction of CHO cellstransfected with rat PACAP receptor protein cDNA (pRPR4-B). The numeralson the abscissa indicate the concentrations (log M) of PACAP27 and VIP,and the numerals on the ordinate indicate the binding (%) of ¹²⁵I!-PACAP27, taking the specific binding as 100, when each peptide isadded so as to give the final concentrations on the abscissa;

FIG. 41 shows the binding of ¹²⁵ I!-PACAP27 in the membrane fraction ofCHO cells transfected with the rat PACAP receptor protein cDNA(pRPR3-A). A indicates CHO cells transfected with a rat PACAP receptorprotein cDNA (pRPR3-A), and B indicates CHO cells transfected with a ratPACAP receptor protein cDNA (pRPR4-B). The numerals on the abscissaindicate sample Nos. of transformant CHO cells obtained by separatingsingle clone-derived colonies, and the numerals on the ordinate indicatethe binding (cpm) of ¹²⁵ I!-PACAP27;

FIG. 42 shows results of examination of reproducibility of clones havingmuch ¹²⁵ I! binding in FIG. 37. The numerals 1 and 2 on the abscissaindicate untreated CHO cells, the numerals 3 and 4 VIP cDNA-introducedCHO cells, the numerals 5 and 6 clone B1, the numerals 7 and 8 clone B2,the numerals 9 and 10 clone B17, the numerals 11 and 12 clone A6, thenumerals 13 and 14 clone A12, and the numerals 15 and 16 clone A15. Thenumerals on the ordinate indicate the binding (cpm) of ¹²⁵ I!-PACAP27;

FIG. 43 are graphs showing the changes in the amounts of intracellularcyclic AMP. The upper graph (type I-A) indicates changes in the amountsof intracellular cyclic AMP of CHO cells transfected with the rat PACAPreceptor protein cDNA (pBPR-T) produced by PACAP27, PACAP38 and VIP. Thenumerals on the abscissa indicate the concentrations (log M) of PACAP27,PACAP38 and VIP, and the numerals on the ordinate indicate theconcentration (ratio (%) to the maximum production amount) ofintracellular cyclic AMP.

The lower graph (type I-B) indicates changes in the amounts ofintracellular cyclic AMP of CHO cells transfected with the rat PACAPreceptor protein cDNA (pBPR-TD) produced by PACAP27, PACAP38 and VIP.The numerals on the abscissa indicate the concentrations (log M) ofPACAP27, PACAP38 and VIP, and the numerals on the ordinate indicate theconcentration (ratio (%) to the maximum production amount) ofintracellular cyclic AMP;

FIG. 44 shows the amount of rat PACAP receptor protein expressed intransformant Sf9 cells with baculoviruses. Sf9 was infected with 10clones of recombinant viruses at the stage of primary plaquemeasurement, and 4 days after culture, the binding of the cells to ¹²⁵I!-PACAP27 was assayed. The numerals on the abscissa indicate sampleNos. of the transformant Sf9 cells. Sample Nos. 1 to 3 indicatetransformant Sf9 cells containing rat PACAP receptor protein cDNAintroduced by a vector modified from pRPR3-A, sample Nos. 4 to 6indicate transformant Sf9 cells containing rat PACAP receptor proteincDNA introduced by a vector modified from pRPR4-B, and sample No. 7indicates uninfected Sf9 cells (control). Cold (-) on the ordinateindicates the binding of each sample to ¹²⁵ I!-PACAP27 when only 100 pM¹²⁵ I!-PACAP27 is added, and cold (+) indicates the binding of eachsample to ¹²⁵ I!-PACAP27 when 100 pM ¹²⁵ I!-PACAP27 and 1 μm unlabeledPACAP27 are added;

FIG. 45 shows the amount of human PACAP receptor protein expressed intransformant Sf9 cells with baculoviruses. Sf9 was infected with 10clones of recombinant viruses at the stage of primary purification, andcultured for 4 days after infection. The binding of ¹²⁵ I!-PACAP27 onthe cells was assayed. The numerals on the abscissa indicate sampleNos., and the numerals on the ordinate indicate the amount of ¹²⁵I!-PACAP27 binding. Sample No. 0 indicates uninfected Sf9 cells(control);

FIG. 46 is a graph showing Scatchard plot in a membrane fraction ofCHO-K1 cells transfected with pTS849 which expresses human PACAPreceptor protein.

FIG. 47 is a graph showing results of the competitive bindingexperiments of PACAP27 (□), PACAP38 (◯) and VIP (Δ) to ¹²⁵ I!-PACAP27 ina membrane fraction of CHO cells transfected with pTS849 which expresseshuman PACAP receptor protein. The numerals on the abscissa indicate theconcentrations (log M) of PACAP27, PACAP38 and VIP, and the numerals onthe ordinate indicate the binding (%) of ¹²⁵ I!-PACAP27, taking thespecific binding as 100, when each peptide is added so as to give thefinal concentrations on the abscissa;

FIG. 48 is a graph showing changes in the amounts of intracellularcyclic AMP of CHO cells transfected with human PACAP receptor proteincDNA (pTS847-1) produced by PACAP27 (□), PACAP38 (◯) and VIP (Δ). Thenumerals on the abscissa indicate the concentrations (log M) of PACAP27,PACAP38 and VIP, and the numerals on the ordinate indicate the relativeconcentrations of cyclic AMP in transformant CHO cells treated withpeptides having respective concentrations, taking the concentration ofinositol phosphate in untreated transformant CHO cells as 1;

FIG. 49 shows results of Northern hybridization using RNA prepared fromthe human brain, lung, liver, thymus, spleen, pancreas and placenta, anda human PACAP receptor protein cDNA probe. The bands represent that theRNA prepared from the human brain, lung, liver, thymus, spleen, pancreasplacenta, and the human PACAP receptor protein cDNA probe exhibit crossreaction. The numerals on the left indicate the size of a molecularweight marker;

FIG. 50 shows results of norther hybridization using RNA prepared fromthe rat olfactory bulbs, amygdalae, cerebral basal ganglia, hippocampi,hypothalami, cerebral cortices, medulla oblongatas, cerebellums,vertebrae and pituitary glands, and a rat PACAP receptor protein cDNAprobe. The bands represent that the RNA prepared from the rat olfactorybulbs, amygdalae, cerebral basal ganglia, hippocampi, hypothalamiccerebral cortices, medulla oblongatas, cerebellums, vertebrae andpituitary glands, and the rat PACAP receptor protein cDNA probe exhibitcross reaction. The numerals on the left indicate the size of amolecular weight marker.

FIGS. 51A-51J show a formula of the compound which was found by thescreening using the membrane fraction of Sf9 cells which expressed humanPACAP receptor Type I-A by baculovirus.

FIG. 52 is a graph which shows a typical sample of screening ofhybridomas.

FIG. 53 shows a detection of PACAP receptor by Western blotting with theantibody of the present invention. Lane 1 shows rainbow coloured proteinmolecular weight markers and lane 2 shows a result of 320 ng of amembrane protein solubilized in an insect cell containing 20 ng of humanPACAP receptor.

FIG. 54 is a graph which shows that the antibody of the presentinvention inhibits binding of PACAP27 and PACAP receptor.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors obtained cDNA clones of the PACAP receptorproteins from the bovine brain cDNA library. Of these, λBPR35, λBPR114and λBPR68 were cloned, and subcloned into pUC118 to obtain pBPR35,pBPR114 and pBPR68 (FIG. 1). Further, pBPR35 and pBPR68 were recombinedat the BamHI site to prepare pBPR-T having a complete translation frame.The complete primary structure of bovine PACAP receptor protein based onpBPR-T was deduced (FIG. 2, pBPR-T). 84 nucleotides were not present inpBPR114, compared with pBPR-T. This is considered to occur byalternative splicing in a transcription product from a common gene.

The PACAP receptor protein which does not contain 84 nucleotides can beprepared by recombinating pBPR-T with pBPR114 at the BamHI and Ava IIsites. The amino acid sequence of the recombinant protein was deduced(FIG. 3, pBPR-TD). The total number of amino acid residues and themolecular weight derived from these sequences are 513 residues (58.5kilodaltons) for pBPR-T, and 485 residues (55.3 kilodaltons) forpBPR-TD. As to both the molecules, the N-terminal sequence up to the37th Ala residue was deduced to be a signal sequence for passing througha membrane.

Further, the N-terminal sequence after this processing completely agreedwith the N-terminal amino acid sequence of bovine PACAP receptor proteinpurified in this invention (FIG. 4). Analysis of hydrophobicity based onthe amino acid sequence revealed in the present invention proved thatbovine PACAP receptor protein has 7 hydrophobic amino acid clustersconsidered to be transmembrane domains in tandem (FIG. 5). Suchstructural features are common to peptide receptor proteins of the Gprotein coupling type European Journal of Pharmacology, 227, 1-8(1992)!.

The present inventors further cloned λRPACAPR18, λRPACAPR46, λRPACAPR5and λRPACAPR12 as cDNA clones of the PACAP protein from the rat braincDNA library, and subcloned into pcDNA I or pUC118 to obtain pRPACAPR18,pRPACAPR46, pRPACAPR5 and pRPACAPR12 (FIG. 6). Further, pRPACAPR46 andpRPACAPR5 were recombined at the BamHI site to prepare pRPACAPR46-5having a complete translation frame. Based on these, two kinds ofcomplete primary structures of rat PACAP receptor protein were deduced(FIGS. 7 and 8, and FIGS. 9 and 10). The first methionine in eachsequence is considered to be an initiation codon. It is conceivable thatthe difference between two kinds of sequences shown in FIGS. 7 and 8 andFIGS. 9 and 10 arises by alternative splicing in a transcription productfrom a common gene. The total number of amino acid residues and themolecular weight derived from these sequences are 467 residues (53.2kilodaltons), and 495 residues (56.4 kilodaltons), respectively. As toboth the molecules, the N-terminal sequence up to the 19th Ala residuewas deduced to be a signal sequence for passing through a membrane.Further, the N-terminal sequence after this processing completely showedhigh homology with the N-terminal amino acid sequence of bovine PACAPreceptor protein purified (FIG. 11).

In addition, the present inventors cloned λ#14 shown in FIG. 12 as acDNA clone of the PACAP protein from the human pituitary cDNA library,and subcloned into pUC118 to obtain pTS847-1. Based on this, thecomplete primary structure of human PACAP receptor protein was deduced(FIG. 13). The first methionine in its sequence is considered to be aninitiation codon. The total number of amino acid residues and themolecular weight derived from FIG. 13 are 525 residues and 59.3kilodaltons, respectively. As to this molecule, the N-terminal sequenceup to the 77th Ala residue was deduced to be a signal sequence forpassing through a membrane. Further, the N-terminal sequence after thisprocessing completely showed high homology with the N-terminal aminoacid sequence of bovine PACAP receptor protein purified by Ohtaki et al(FIG. 14).

The present inventors found out that 84 nucleotides were inserted at thesame sites of both rat and bovine Type I-B of PACAP receptor, andtherefore deduced that there would also be a similar insertion at thesame site of a human PACAP receptor. The inventors prepared a primerfrom the sequence flanking to the deduced insertion site and conductedPCR methods. As a result, the present inventors succeeded in cloning acDNA encoding the insertion region of subtype of a human PACAP receptorand in identifying the amino acid sequence of the insertion region, byapplying PCR method to cDNAs of human pituitay and amygdaloid nucleus.The present inventors further succeeded in preparing a cDNA encodingwhole length of subtype of a human PACAP receptor by recombinating theabove cDNAs with the cDNA of Type I-A of a human PACAP receptor. In moredetail, the present inventors obtained pHPR15A, pHPR55A and pHPR66P ascDNA clones of the insertion region of a subtype of a human PACAPreceptor by applying PCR method to cDNAs of human amygdaloid nucleus andhuman pituitary (FIG. 15). By recombinating the clones with human PACAPreceptor Type I-A at the recognition sites of HpaI and AvaII, a cDNA foreach subtype was constructed. The nucleotide sequences of cDNAs of theconstructed subtypes and the amino acid sequence deduced therefrom areshown in FIGS. 16 to 18.

Analysis of hydrophobicity based on the amino acid sequence revealed inthe present invention proved that rat PACAP receptor protein has 7hydrophobic amino acid clusters considered to be membrane permeabledomains in tandem (FIG. 19(A) indicates a result of analysis of theprotein shown in FIGS. 7 and 8, and FIG. 19(B) indicates a result ofanalysis of the protein shown in FIGS. 9 and 10). Such structuralfeatures are common to peptide receptor proteins of the G proteinbinding type European Journal of Pharmacology, 227, 1-8 (1992)!. Thepeptides or ligands were extremely similar in structure, and the resultof comparison at the amino acid sequence level with the structure of aVIP receptor used as a cDNA probe for cloning revealed extremely highsimilarity. As a whole, the similarity of the N-terminal portions isvery low, whereas regions containing the first to seventh membranepermeable domains and the C-terminal intracellular domains converselyshowed high similarity. It was further revealed that human PACAPreceptor protein also has 7 hydrophobic amino acid clusters consideredto be membrane permeable domains in tandem (FIG. 21).

The amino acid sequences of bovine PACAP receptor protein, rat PACAPreceptor protein and human PACAP receptor protein of the presentinvention showed very high homology (FIG. 22). All of these proteinswere proved to shave amino acid sequences represented by SEQ ID NOs: 1to 12.

The present inventors named rat PACAP receptor protein having the aminoacid sequence of SEQ ID NO: 19 "Type I-A", and rat PACAP receptorprotein having the amino acid sequence of SEQ ID NO: 21 "Type I-B".Bovine PACAP receptor protein having the amino acid sequence of SEQ IDNO: 15 is bovine PACAP receptor protein Type I-A corresponding to ratPACAP receptor protein Type I-A of SEQ ID NO: 19, and bovine PACAPreceptor protein having the amino acid sequence of SEQ ID NO: 17 isbovine PACAP receptor protein Type I-B corresponding to rat PACAPreceptor protein Type I-B of SEQ ID NO: 21.

Human PACAP receptor protein having the amino acid sequence of SEQ IDNO: 23 is human PACAP receptor protein Type I-A corresponding to ratPACAP receptor protein Type I-A of SEQ ID NO: 19, and human PACAPreceptor protein having the amino acid sequence of SEQ ID NO: 25 whichclone is obtained by recombinating pHPR15A is human PACAP receptorprotein Type I-B. pHPR55A lacks 3 nucleotides, CAG, from pHPR15A, whichlacks Ser as an amino acid. The human PACAP receptor protein having anamino acid sequence of SEQ ID NO:27 was named "Type I-B2" since theprotein is thought to be a clone resulting from a sliding of theposition of a splicing of Type I-B. Further, a human PACAP receptorprotein having an amino acid sequence of SEQ ID NO:29, a recombinantclone of pHPR66P, which is thought to result from a transcriptionproduct of a common gene by an alternative splicing and the subtype wasnamed Type I-C.

The origin of amino acid sequences of PACAP receptor proteins andnucleotide sequences of DNAs coding for said proteins represented by SEQID NO used in this specification are as follows:

SEQ ID NO: 1-SEQ ID NO: 12!

These indicate amino acid sequences which bovine, rat and human PACAPreceptor proteins have in common;

SEQ ID NO: 13!

This indicates an N-terminal amino acid sequence of the purified bovinePACAP receptor protein;

SEQ ID NO: 14!

This indicates an amino acid sequence of a protein in which a signalpeptide is deleted from bovine PACAP receptor protein Type I-A encodedby in pBPR-T;

SEQ ID NO: 15!

This indicates an amino acid sequence of bovine PACAP receptor proteinType I-A encoded by in pBPR-T;

SEQ ID NO: 16!

This indicates an amino acid sequence of a protein in which a signalpeptide is deleted from bovine PACAP receptor protein Type I-B encodedby in pBPR-TD;

SEQ ID NO: 17!

This indicates an amino acid sequence of bovine PACAP receptor proteinType I-B encoded by in pBPR-TD;

SEQ ID NO: 18!

This indicates an amino acid sequence of a protein in which a signalpeptide is deleted from rat PACAP receptor protein Type I-A;

SEQ ID NO: 19!

This indicates an amino acid sequence of rat PACAP receptor protein TypeI-A;

SEQ ID NO: 20!

This indicates an amino acid sequence of a protein in which a signalpeptide is deleted from rat PACAP receptor protein Type I-B;

SEQ ID NO: 21!

This indicates an amino acid sequence of rat PACAP receptor protein TypeI-B;

SEQ ID NO: 22!

This indicates an amino acid sequence of a protein in which a signalpeptide is deleted from human PACAP receptor protein Type I-A;

SEQ ID NO: 23!

This indicates an amino acid sequence of human PACAP receptor proteinType I-A;

SEQ ID NO: 24!

This indicates an amino acid sequence of a protein in which a signalpeptide is deleted from human PACAP receptor protein Type I-B;

SEQ ID NO: 25!

This indicates an amino acid sequence of human PACAP receptor proteinType I-B;

SEQ ID NO: 26!

This indicates an amino acid sequence of a protein in which a signalpeptide is deleted from human PACAP receptor protein Type I-B2;

SEQ ID NO: 27!

This indicates an amino acid sequence of human PACAP receptor proteinType I-B2;

SEQ ID NO: 28!

This indicates an amino acid sequence of a protein in which a signalpeptide is deleted from human PACAP receptor protein Type I-C;

SEQ ID NO: 29!

This indicates an amino acid sequence of human PACAP receptor proteinType I-C;

SEQ ID NO: 30!

This indicates a nucleotide sequence of cDNA coding for bovine PACAPreceptor protein Type I-A;.

SEQ ID NO: 31!

This indicates a nucleotide sequence of cDNA coding for bovine PACAPreceptor protein Type I-B;

SEQ ID NO: 32!

This indicates a nucleotide sequence of cDNA coding for rat PACAPreceptor protein Type I-A;

SEQ ID NO: 33!

This indicates a nucleotide sequence of cDNA coding for rat PACAPreceptor protein Type I-B;

SEQ ID NO: 34!

This indicates a nucleotide sequence of cDNA coding for human PACAPreceptor protein Type I-A;

SEQ ID NO: 35!

This indicates a nucleotide sequence of cDNA coding for human PACAPreceptor protein Type I-B;

SEQ ID NO: 36!

This indicates a nucleotide sequence of cDNA coding for human PACAPreceptor protein Type I-B2;

SEQ ID NO: 37!

This indicates a nucleotide sequence of cDNA coding for human PACAPreceptor protein Type I-C;

SEQ ID NO: 38!

This indicates a nucleotide sequence of DNA (pBPR-T) containing anucleotide sequence of cDNA coding for bovine PACAP receptor proteinType I-A;

SEQ ID NO: 39!

This indicates a nucleotide sequence of DNA (pBPR-TD) containing anucleotide sequence of cDNA coding for bovine PACAP receptor proteinType I-B;

SEQ ID NO: 40!

This indicates a nucleotide sequence of DNA (PRPACAPR 46-5) containing anucleotide sequence of cDNA coding for rat PACAP receptor protein TypeI-A;

SEQ ID NO: 41!

This indicates a nucleotide sequence of DNA (pRPACAPR 12) containing anucleotide sequence of cDNA coding for rat PACAP receptor protein TypeI-B;

SEQ ID NO: 42!

This indicates a nucleotide sequence of DNA (pTS847-1) containing anucleotide sequence of cDNA coding for human PACAP receptor protein TypeI-A;

SEQ ID NO: 43!

This indicates a nucleotide sequence of DNA containing a nucleotidesequence of cDNA coding for human PACAP receptor protein Type I-B;

SEQ ID NO: 44!

This indicates a nucleotide sequence of DNA containing a nucleotidesequence of cDNA coding for human PACAP receptor protein Type I-B2;

SEQ ID NO: 45!

This indicates a nucleotide sequence of DNA containing a nucleotidesequence of cDNA coding for human PACAP receptor protein Type I-C;

SEQ ID NO: 46!

This indicates an amino acid sequence of PACAP38.

SEQ ID NO: 47!

This indicates an amino acid sequence of PACAP27.

SEQ ID NO: 48!

This indicates a nucleotide sequence of an oligonucleotide used forscreening of cDNA coding for rat PACAP receptor proteins Type I-A andType I-B;

SEQ ID NO: 49!

This indicates a nucleotide sequence of an oligonucleotide used forscreening of cDNA coding for rat PACAP receptor proteins Type I-A andType I-B.

SEQ ID NO: 50!

This indicates an N-terminal amino acid sequence (sequence consisting of16 amino acids) of bovine PACAP receptor protein.

SEQ ID NO: 51!

This indicates a nucleotide sequence of an oligonucleotide used forscreening of cDNA encoding bovine and human PACAP receptor proteins.

SEQ ID NO: 52!

This indicates a nucleotide sequence of a primer prepared from cDNAencoding human PACAP receptor protein Type I-A.

SEQ ID NO: 53!

This indicates a nucleotide sequence of a primer prepared from cDNAencoding human PACAP receptor protein Type I-A.

SEQ ID NO: 54!

This indicates a nucleotide sequence of a probe prepared based on thenucleotide sequence at the insertion region of rat PACAP receptorprotein Type I-B.

SEQ ID NO: 55!

This indicates a nucleotide sequence of a probe prepared based on thenucleotide sequence at other insertion region than rat PACAP receptorprotein Type I-B.

When nucleotides, amino acids and so on are indicated by abbreviationsin the specification and drawings, the abbreviations adopted by theIUPAC-IUB Commission on Biochemical Nomenclature or commonly used in theart are employed. For example, the following abbreviations are used.When the amino acids are capable of existing as optical isomers, it isunderstood that the L-forms are represented unless otherwise specified.

DNA: Deoxyribonucleic acid

cDNA: Complementary deoxyribonucleic acid

A: Adenine

T: Thymine

G: Guanine

C: Cytosine

RNA: Ribonucleic acid

mRNA: Messenger ribonucleic acid

dATP: Deoxyadenosine triphosphate

dTTP: Deoxythymidine triphosphate

dGTP: Deoxyguanosine triphosphate

dCTP: Deoxycytidine triphosphate

ATP: Adenosine triphosphate

EDTA: Ethylenediaminetetraacetic acid

SDS: Sodium dodecyl sulfate

EIA: Enzyme immunoassay

Gly: Glycine

Ala: Alanine

Val: Valine

Leu: Leucine

Ile: Isoleucine

Ser: Serine

Thr: Threonine

Cys: Cysteine

Met: Methionine

Glu: Glutamic acid

Asp: Aspartic acid

Lys: Lysine

Arg: Arginine

His: Histidine

Phe: Phenylalanine

Tyr: Tyrosine

Trp: Tryptophan

Pro: Proline

Asn: Asparagine

Gin: Glutamine

Further, meanings of other abbreviations used in this specification areas follows:

VIP: Vasoactive intestinal peptide

Tris: Tris(hydroxymethyl)aminomethane

EDTA: Ethylenediaminetetraacetic acid

PMSF: Phenylmethylsulfonyl fluoride

BSA: Bovine serum albumin

CHAPS: 3- (3-Cholamidopropyl)dimethylammonio!-1-propanesulfonate

Biotin-HSDP: N- 6-(biotinamido)hexyl!-3',-(2'-pyridylthio)-propionicacid amide

"TM" used in this specification represents a registered trade mark.

The PACAP receptor proteins which are capable of binding a PACAP of thepresent invention may be derived from tissues of warm-blooded animals(for example, the cerebrums, pituitary glands and adrenals of rats,mice, hamsters, chickens, dogs, cats, sheep, monkeys, pigs, cattle orhumans) or cells for example, adrenal chromaffin cells, glial cells andestablished cell lines (such as PC12 cells, NB-OK cells and AR4-2Jcells)!, or may be produced by chemical synthesis. Any proteins may beused as long as they have PACAP receptor activity ("PACAP receptoractivity" means the action of specifically binding to the PACAPs).Examples thereof include proteins having amino acid sequences containingat least one member selected from the group consisting of the amino acidsequences represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. Such proteinsinclude, for example, proteins having only the amino acid sequences ofSEQ ID NO: 1 to SEQ ID NO: 12, respectively, and proteins in which aminoacids and (or) peptides are further bound to said proteins at theirN-terminal sites and (or) C-terminal sites. Preferable examples of suchproteins include proteins having amino acid sequences containing theamino acid sequences represented by SEQ ID NO:1 to SEQ ID NO:12.Specifically, they include proteins having the amino acid sequencesrepresented by SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29. Preferably, the proteinshaving the amino acid sequences represented by SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16 or SEQ ID NO: 17 are bovine-derived proteins, theproteins having the amino acid sequences represented by SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 are rat-derived proteins,and the proteins having the amino acid sequences represented by SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29 are human derived proteins.

The amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 areamino acid sequences which the bovine-, rat- and human-derived PACAPreceptors which lack signal peptide, that is PACAP receptors having theamino acid sequences represented by SEQ ID NO: 14, 16, 18, 20, 22, 24,26 or 28, have in common (FIG. 22). Further, as apparent from FIG. 22,the amino acid sequences of the PACAP receptor proteins exhibit highhomology among species of warm-blooded animals, so that proteins havingusually 90-100%, preferably 95-100%, and more preferably 97-100%homology with the amino acid sequence(s) represented by SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 26 and/or SEQ ID NO: 28 are also included in the PACAPreceptor proteins of the present invention.

Further, for example, proteins having amino acid sequences containingthe amino acid sequence represented by SEQ ID NO: 13 can also be used.Examples of such proteins include proteins having only the amino acidsequence of SEQ ID NO: 13, and proteins in which amino acids or peptidesare further bound to said proteins at their C-terminal sites.Specifically, proteins having the amino acid sequence of SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 are used. In particular,bovine-derived proteins are preferred.

Furthermore, the PACAP receptor proteins of the present invention alsocomprise proteins in which the N-terminal Met residues are protectedwith protective groups (for example, C₁₋₆ acyl groups such as formyl andacetyl), proteins in which peptide bond between the 9th Lys residues andthe 10th Glu residues in the amino acid sequences represented by SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 are cleaved in vivo and theGlu residues are pyroglutaminated, proteins in which side chains ofamino acids in molecules are protected with appropriate protectivegroups, and conjugated proteins such as so-called glicoproteins to whichsugar chains are bound.

As used herein, "PACAP receptor protein" also includes a salt of saidprotein. Salts of the PACAP receptor proteins used in the presentinvention include, for example, salts with inorganic acids (such ashydrochloric acid, phosphoric acid, hydrobromic acid and sulfuric acid)and salts with organic acids (such as acetic acid, formic acid,propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid,citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acidand benzenesulfonic acid).

In producing the proteins of the present invention, when the proteinsare extracted from animal tissues or cells, methods for purifyingproteins can generally be employed. In particular, the proteins of thepresent invention are of the membrane binding type, so thatsolubilization of membrane fractions is required. Concrete purifyingmethods are shown below:

(1) Preparation of a membrane fraction suspension

A membrane fraction suspension can be prepared by treating an animaltissue, for example, by the method described in Biochem. Biophys. Res.Commun., 172, 709-714 (1990) or a method based thereon.

(2) Solubilization of a desired protein fraction from the membranefraction suspension

The membrane fraction obtained in (1) described above is solubilized bythe method described in Biochem. Biophys. Res. Commun., 172, 709-714(1990) or a method based thereon. Examples of solubilizing reagentswhich can be used therein include detergents having skeletons of bileacid (such as digitonin and CHAPS) and nonionic detergents (such asTWEEN 20™ and TRITON-X™). Specifically, the membrane fraction suspensionis diluted with an appropriate buffer (for example, Tris buffer) to givea protein concentration of 0.1 to 5.0 mg/ml, preferably 0.5 to 2.0mg/ml, and more preferably 1.0 mg/ml, the above-mentioned solubilizingreagent is added thereto to yield a concentration of 0.1 to 5.0%,preferably 0.5 to 2.0%, and more preferably 1.0%, and the mixture isstirred usually for 10 minutes to 72 hours, preferably for 30 minutes to24 hours, followed by ultracentrifugation to obtain a supernatantthereof. The presence or absence of a desired protein can be detected bymeasuring the activity of said protein. For example, PACAP receptoractivity can be supernatant thereof. The presence or absence of adesired protein can be detected by measuring the activity of saidprotein. For example, PACAP receptor activity can be measured by themethod described in Biochem. Biophys. Res. Commun., 172, 709-714 (1990)or methods based thereon.

(3) Purification of the desired protein from the solubilized sample

Purification of the desired protein from the solubilized sample obtainedin (2) described above can be conducted by anion-exchange chromatographyfor example, DEAE-TOYOPEARL™ (Tosoh)!, hydroxyapatite chromatography forexample, HCA-100™ (Mitsui Toatsu Chemicals)!, affinity chromatographyfor example, avidin-agarose (Pierce)!, gel filtration for example,SUPERROSE™ (Pharmacia)!, etc. under appropriate conditions. Inparticular, the methods for producing the desired protein in the presentinvention are characterized in that the desired protein can be purifiedat high efficiency by use of affinity chromatography using the"biotinylated PACAPs" first discovered as ligands in the presentinvention. As said PACAPs, PACAP27 to PACAP38 described in EP-A-0404652and PACAP23 to PACAP26 described in EP-A-0467279 are used. Inparticular, PACAP27 and PACAP38 are preferred. Examples of methods forbiotinylating the PACAP include the method of introducing a cysteineresidue into the carboxyl terminus of the PACAP to synthesize a PACAPderivative, and easily binding a commercially available biotinylatingreagent through the cysteine residue. As the derivatives, for example,PACAP27-Cys and PACAP38-Cys are used. The derivatives can be produced bymethods known in the art or methods based thereon, for example, solidphase methods. The biotinylating reagents used include, for example, thefollowing reagents: ##STR3## N-6-(Biotinamido)hexyl!-3'-(2'-pyridylthio)-propionic acid amide (Cat. No.21341, Pias) ##STR4## 6-N'{2-(N-Maleimido)ethyl}-N-piperazinylamido!hexyl biotinamide (Code No.344-06391, Dojin Kagaku Kenkyusho)

The binding of the derivatives to the biotinylating reagents can becarried out by the method described in Biochem. Biophys. Res. Commun.,184, 123-160 (1990) or methods based thereon.

Examples of the biotinated PACAPs of the present invention include onesrepresented by the following formula: ##STR5##

In particular, one represented by ##STR6## are preferred.

Of these biotinylated PACAPs, the method for producing biotinylatedPACAP27 is described in detail in Example 1 (5) set out below. Otherligands can also be produced in accordance with the method of Example 1(5-1).

The biotinylated PACAPs of the present invention thus obtained havebinding ability to both PACAP receptor proteins and avidin. They can betherefore used for many purposes such as staining and flowcytophotometry of cells and tissues, as well as purification of PACAPreceptor proteins.

On the other hand, when the proteins of the present invention areproduced by chemical synthesis, they are produced by methods known inthe art or methods based thereon. For example, either solid phasesynthesis methods or liquid phase synthesis methods may be employed.Namely, the desired peptides can be produced by condensing partialpeptides or amino acids which can constitute the proteins of the presentinvention with residual moieties, and eliminating protective groups whenthe products have the protective groups. Known condensing methods andelimination of the protective groups include, for example, methodsdescribed in (1) to (5) given below:

(1) M. Bodansky and M. A. Ondetti, Peptide Synthesis, IntersciencePublishers, New York (1966);

(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965);

(3) N. Izumiya et al., Peptide Gosei no Kiso to Jikken (Fundamentals andExperiments of Peptide Synthesis), Maruzen (1985);

(4) H. Yazima, S. Sakakibara et al., Seikagaku Jikken Koza (Course ofBiochemical Experiments), 1, Chemistry of Proteins IV, 205 (1977); and

(5) Zoku Iyakuhin no Kaihatu (Development of Drugs) second series), 14,Peptide Synthesis, supervised by H. Yazima, Hirokawa Shoten.

After reaction, the PACAP receptor proteins of the present invention canbe isolated by combinations of usual purification methods such assolvent extraction, distillation, reprecipitation, columnchromatography, liquid chromatography, and recrystallization.

When the PACAP receptor proteins obtained by the above-mentioned methodsare free forms, they can be converted to appropriate salts by knownmethods. Conversely, when the proteins are obtained in the salt state,they can be converted to the free forms by known methods.

The receptor fragments capable of binding a PACAP of the presentinvention may be any peptides e.g., a receptor fragment or a truncatedreceptor, as long as they have PACAP receptor activity. For example,sites of PACAP receptor protein molecules exposed out of cell membranesare used. Specifically, they are partial peptides deduced to beextracellular regions in hydrophobic plot analysis (FIG. 23 to FIG. 27).Examples thereof include:

(1) peptides having the amino acid sequence consisting of the 38th to164th, 223rd to 232nd, 303rd to 317th or 416th to 424th amino acidresidues of SEQ ID NO: 15 (bovine PACAP receptor protein Type I-A) (FIG.23);

(2) peptides having the amino acid sequence consisting of the 38th to164th, 223rd to 232nd, 303rd to 317th or 388th to 397th amino acidresidues of SEQ ID NO: 17 (bovine PACAP receptor protein Type I-B) (FIG.24);

(3) peptides having the amino acid sequence consisting of the 20th to146th, 205th to 214th, 286th to 299th or 369th to 378th amino acidresidues of SEQ ID NO: 19 (rat PACAP receptor protein Type I-A) (FIG.25);

(4) peptides having the amino acid sequence consisting of the 20th to146th, 205th to 214th, 286th to 299th or 397th to 406th amino acidresidues of SEQ ID NO: 21 (rat PACAP receptor protein Type I-B) (FIG.26); and

(5) peptides having the amino acid sequence consisting of the 78th to204th, 263rd to 272nd, 342nd to 357th or 427th to 436th amino acidresidues of SEQ ID NO: 23 (human PACAP receptor protein Type I-A) (FIG.27).

The receptor fragments capable of binding a PACAP can be produced byknown methods for synthesizing the peptides of (1) to (5) describedabove or by cleaving the PACAP receptor proteins with appropriatepeptidases.

Salts of the receptor fragments capable of binding a PACAP used in thepresent invention include, for example, salts with inorganic acids (suchas hydrochloric acid, phosphoric acid, hydrobromic acid and sulfuricacid) and salts with organic acids (such as acetic acid, formic acid,propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid,citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acidand benzenesulfonic acid).

The DNAs coding for the PACAP receptor proteins of the present inventionmay be any, as long as they have nucleotide sequences coding for thePACAP receptor proteins. Namely, the DNAs encoding the PACAP receptorproteins of the present invention may be any of cDNA, genomic DNA andsynthetic DNA. Further, the DNAs may be ones encoding the PACAP receptorproteins derived from any warm-blooded animals (for example, rats, mice,hamsters, chickens, dogs, cats, sheep, monkeys, pigs, cattle, humans andso on), namely the above-mentioned PACAP receptor proteins of thepresent invention. Specifically, the DNAs having the nucleotidesequences of SEQ ID NO: 30 to SEQ ID NO: 45, respectively, are used.Screening of the DNAs can be conducted by general genetic engineeringtechniques or methods based thereon, for example, based on the examples2 to 4 given below.

Expression vectors for the PACAP receptor proteins can be produced by(a) restricting desired DNA fragments from the DNAs encoding the PACAPreceptor proteins, and (b) ligating the DNA fragments downstream frompromoters in appropriate vectors.

The cloned DNAs encoding the PACAP receptor proteins can be used assuch, or after digestion with restriction enzymes or addition of linkersif desired, depending on their purpose.

The DNA may have ATG as a translation initiation codon on the5'-terminal side, and TAA, TGA or TAG as a translation termination codonon the 3'-terminal side. The translation initiation codon andtranslation termination codon may be added by use of an appropriatesynthetic DNA adaptor. A promoter is further ligated upstream therefromto express the DNA.

The vectors include plasmids derived from Escherichia coli (for example,pBR322, pBR325, pUC12 and pUC13), plasmids derived from Bacillussubtilis (for example, pUB110, pTP5 and pC194), plasmids derived fromyeast (for example, pSH19 and pSH15, bacteriophages (for example, λphage), and viruses such as retroviruses, vaccinia viruses andbaculoviruses.

As the promoter used in the present invention, any promoter is availableas long as it is suitable for expression corresponding to a host cellused for the gene expression.

When the host cell used for transformation is Escherichia, a trppromoter, a lac promoter, a recA promoter, a λP_(L) promoter or an lpppromoter is preferred. When the host cell is Bacillus, an SPO1 promoter,an SPO2 promoter or a penP promoter is preferred. When the host cell isyeast, a PHO5 promoter, a PGK promoter, a GAP promoter or an ADHpromoter is preferred.

When the host cell is an animal cell, a SV40-derived promoter, aCMV-derived promoter, a retrovirus promoter, a metallothionein promoter,etc. are each usable.

An enhancer is also effectively utilized for expression.

Using the vectors containing the DNAs coding for the PACAP receptorproteins thus constructed, transformants are prepared.

Examples of the host cells include Escherichia, Bacillus, yeast, insectsand animal cells.

Examples of the above-mentioned Escherichia include E. coli K12.DH1Proc. Natl. Acad. Sci. U.S.A., 60, 160 (1968)!, JM103 Nucleic AcidsResearch, 9, 309 (1981)!, JA221 Journal of Molecular Biology, 120, 517,(1978)!, HB101 Journal of Molecular Biology, 41, 459 (1969)! and C600Genetics, 39, 440 (1954)!.

Examples of the above-mentioned Bacillus include Bacillus subtilis M1114Gene, 24, 255 (1983)! and 207-21 Journal of Biochemistry, 95, 87(1984)!.

Examples of the above-mentioned yeast include Saccharomyces cerevisiaeAH22, AH22R⁻, NA87-11A, DKD-5D and 20B-12.

Examples of the insects include larvae of silk worms Maeda et al.,Nature, 315, 592 (1985)!.

Examples of the animal cells include monkey cell COS-7, Vero, Chinesehamster cell (CHO), mouse L cell and human FL cell.

The transformation of the above-mentioned Escherichia is conducted, forexample, according to the method described in Proc. Natl. Acad. Sci.U.S.A., 69, 2110 (1972), Gene, 17, 107 (1982) or the like.

The transformation of the Bacillus is carried out, for example,according to the method described in Molecular & General Genetics, 168,111 (1979) or the like.

The transformation of the yeast is performed, for example, according tothe method described in Proc. Natl. Acad. Sci. U.S.A., 75, 1929 (1978).

The transformation of the insects is conducted, for example, accordingto the method described in Bio/Technology, 6, 47-55 (1988) or the like.

The transformation of the animal cells is carried out, for example,according to the method described in Virology, 52, 456 (1973).

Thus, the transformants transformed with the expression vectorscontaining the cDNAs coding for the PACAP receptor proteins areobtained.

When the bacterial transformants are cultivated, a liquid medium istypically used for cultivation. Carbon sources, nitrogen sources,inorganic compounds and other nutrients necessary for growth of thetransformants are contained therein. Examples of the carbon sourcesinclude glucose, dextrin, soluble starch and sucrose. Examples of thenitrogen sources include inorganic or organic materials such as ammoniumsalts, nitrates, corn steep liquor, peptone, casein, meat extracts,soybean meal and potato extract solution. The inorganic compoundsinclude, for example, calcium chloride, sodium dihydrogenphosphate andmagnesium chloride. Yeast, vitamins and growth promoting factors, etc.may be further added.

The pH of the medium is preferably about 5 to about 8.

When the Escherichia transformants are cultivated, M9 medium containingglucose and Casamino Acids Miller, Journal of Experiments in MolecularGenetics, 431-433, Cold Spring Harbor Laboratory, New York (1972)! ispreferably used to cultivate the transformants. In order to allow thepromoters to act more efficiently, for example, drugs such as 3β-indolylacrylic acid may be added thereto if necessary.

The Escherichia transformants are usually cultivated at about 15° to 43°C. for about 3 to 24 hours with aeration or agitation if necessary.

The Bacillus transformants are usually cultivated at about 30° to 40° C.for about 6 to 24 hours with aeration or agitation if necessary.

When the yeast transformants are cultivated, a preferred medium isBurkholder minimum medium K. L. Bostian, Proc. Natl. Acad. Sci. U.S.A.,77, 4505 (1980)! or SD medium containing 0.5% Casamino Acids G. A.Bitter et al., Proc. Natl. Acad. Sci. U.S.A., 81, 5330 (1984)!. The pHof the medium is preferably adjusted to about 5 to 8. The cultivation isusually carried out at about 20° to 35° C. for about 24 to 72 hours withaeration or agitation if necessary.

When the insect transformants are cultivated, examples of media usedinclude Grace's insect medium (T. C. C. Grace, Nature, 195, 788 (1962)!supplemented with an additive such as 10% inactivated bovine serum. ThepH of the medium is preferably adjusted to about 6.2 to 6.4. Thecultivation is usually carried out at about 27° C. for about 3 to 5 dayswith aeration or agitation if necessary.

When the animal cell transformants are cultured, examples of media usedinclude MEM medium containing about 5 to 20% fetal calf serum (Science,122, 501 (1952)!, DMEM medium Virology, 8, 396 (1959)!, RPMI 1640 mediumJournal of the American Medical Association, 199, 519 (1967)! and 199medium Proceeding of the Society for the Biological Medicine, 73, 1(1950)!. The pH is preferably about 6 to 8. The cell culture is usuallycarried out at about 30° to 40° C. for about 15 to 60 hours, withaeration or agitation if necessary.

The isolation and purification of the PACAP receptor proteins from theabove-mentioned culture products can be carried out, for example,according to the following method.

When the PACAP receptor protein is to be extracted from cultured cells,the cells are collected by methods known in the art after cultivation.Then, the collected cells are suspended in an appropriate buffersolution, and disrupted by ultrasonic treatment, lysozyme treatmentand/or freeze-thawing thereby releasing the PACAP receptor protein,followed by centrifugation to obtain a crude extract of the PACAPreceptor protein. The buffer solution may contain a protein denaturantsuch as urea or guanidine hydrochloride, or a detergent such as TritonX-100.

When the PACAP receptor protein is secreted in the culture solution, asupernatant is separated from the cells by methods known in the artafter termination of cultivation, and then collected. The separation andpurification of the PACAP receptor protein contained in the culturesupernatant or the extract thus obtained can be carried out byappropriate combinations of well-known separating and purifying methods.These known separating and purifying methods include methods utilizing adifference in solubility such as salting-out and solvent precipitation,methods mainly utilizing a difference in molecular weight such asdialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gelelectrophoresis, methods utilizing a difference in electric charge suchas ion-exchange chromatography, methods utilizing specific affinity suchas affinity chromatography, methods utilizing a difference inhydrophobicity such as reverse phase high performance liquidchromatography, and methods utilizing a difference in isoelectric pointsuch as isoelectric point electrophoresis.

Before or after purification, an appropriate protein modifying enzymecan also be reacted with the PACAP receptor protein produced by arecombinant to arbitrarily modify the protein or to partially eliminatea polypeptide therefrom. The protein modifying enzymes used includetrypsin, chymotrypsin, arginyl endopeptidase and protein kinase.

The activity of the PACAP receptor proteins thus obtained can bemeasured by enzyme immunoassays. When the products havedephosphorylation activity, the measurement can also be conducted basedupon said activity.

In the PACAP receptor proteins of the present invention, the amino acidsequences thereof may be partially modified (addition, elimination orsubstitution with other amino acids).

The PACAP receptor proteins and the DNAs coding for said proteins of thepresent invention thus obtained can be used for (i) acquisition ofantibodies and antisera, (ii) construction of expression systems ofrecombinant receptor proteins, (iii) development of receptor bindingassay preparation of probes and PCR primers in gene diagnosis, and (vi)detection of PACAPs or PACAP receptors in vivo. In particular, theinformation hitherto obtained suggests that the PACAPs are deeplyrelated to the functions of the hypothalamus-pituitary gland system, thesympathetic nerve system and the central nerve system. Accordingly,elucidation of the structure and properties of the PACAP receptors cancontribute to development of unique drugs acting on these systems.

The PACAP receptor proteins, the PACAP receptor fragments and the DNAsencoding said proteins of the present invention can be used as follows(1) to (3)

(1) The PACAPs are known to exhibit functions such as protection ofnerve cells and growth maintenance of the nerve cells in vivo. Adecrease in PACAP concentration in vivo is therefore considered toinduce death of the nerve cells and to cause neuropathy such asAlzheimer's disease. Accordingly, for the PACAP receptor proteins of thepresent invention which specifically react with the PACAPS, the partialpeptides thereof or the salts thereof, the PACAP concentration in vivocan be determined high sensitively, so that they can be effectively usedas diagnostic composition for neuropathy such as Alzheimer's disease.When the PACAP receptor proteins of the present invention, the partialpeptides thereof or the salts thereof are used as diagnostic compositionwhich can determine the PACAP composition for neuropathy such asAlzheimer's disease. The diagnosis can be conducted by determining anamount of PACAP which binds to PACAP receptor proteins, the partialpeptides thereof or the salts thereof of the present invention whencontacting the test sample with PACAP receptor proteins, the partialpeptides thereof of the salts thereof of the present invention. When thePACAP receptor proteins of the present invention, the partial peptidesthereof of the salts thereof are used as diagnostic composition whichcan determine the PACAP concentration in test samples, they can be used,for example, in combination with competitive assays. For example, themethods described in the following (i) or (ii), or methods based thereoncan be used:

(i) Radioimmunoassay, edited by H. Irie, Kodansha (1974), and

(ii) Radioimmunoassay (second series), edited by H. Irie, Kodansha(1979)

Specifically, standard curves can be prepared by the receptorcompetitive binding experiment method described in Example 1 (3) givenlater, thereby measuring the PACAP concentration in test samples. Theprocedure of the method is shown below.

                  TABLE 1    ______________________________________     ##STR7##     ##STR8##     ##STR9##     ##STR10##     ##STR11##     ##STR12##     ##STR13##     ##STR14##     ##STR15##    ______________________________________

(2) In the case of a patient suffering from neuropathy (for example,Alzheimer's disease) in which the PACAP action is not sufficientlyexhibited, because the PACAP can not be bound to the PACAP receptor invivo due to a reduction in the amount of the PACAP receptor protein onthe nerve cell membranes in vivo, causing the tendency of death of thenerve cells, the amount of the PACAP receptor protein in the nerve cellsof the patient can be increased by (a) inserting the DNA of the presentinvention in the patient to express it, or by (b) inserting the DNA ofthe present invention in the nerve cells to express it, followed byimplantation of the nerve cells in the patient, thereby sufficientlyexhibiting the PACAP action. That is, the DNAs of the present inventioncan be used for gene therapy of neuropathy, because we can transformnerve cells in vitro or in vivo by using the DNAs of the presentinvention.

The above-mentioned gene therapy can be given according to methods knownin the art. For example, they can be given orally as tablets, capsules,elixirs and microcpsules, or parenterally in the form of injections suchas sterile solutions or suspensions with water or with pharmaceuticallyacceptable solutions other than water. For example, the DNAs of thepresent invention can be mixed with carriers, flavoring agents,excipients, vehicles, preservatives, stabilizing agents, binders, etc.in the form of unit dosage required for generally admittedpharmaceutical practice to prepare preparations. The amount of activeingredients in these preparations is adjusted so as to obtainappropriate doses within specified ranges. Additives which can be mixedwith tablets, capsules, etc. include, for example, binders such asgelatin, corn starch, tragacanth and gum arabic; excipients such ascrystalline cellulose; swelling agents such as corn starch, gelatin andalginic acid; lubricants such as magnesium stearate; sweeteners such assucrose, lactose and saccharine; and flavoring agents such aspeppermint, acamono oil and cherry. When the preparation unit is in thecapsule form, liquid carriers such as fat and oil may further be addedto materials of the above-mentioned types. Sterile compositions forinjection can be formulated according to usual pharmaceutical practicesuch as dissolution or suspension of active substances and naturallyoccurring vegetable oils such as sesame oil and coconut oil in vehiclessuch as water for injection. Aqueous solutions for injection includephysiological saline and isotonic solutions containing glucose or otheradjuvants (for example, D-sorbitol, D-mannitol and sodium chloride), andmay be used in combination with appropriate solubilizing adjuvants suchas alcohols (for example, ethanol), polyalcohols (for example,polypropylene glycol and polyethylene glycol) and nonionic surfaceactive agents (for example, Polysolvate 80 and HCO-50). Oily solutionsinclude sesame oil and soybean oil, and may be used in combination withsolubilizing adjuvants such as benzyl benzoate, benzyl alcohol, etc. Thepreparations may further contain buffers (for example, phosphate bufferand sodium acetate buffer), soothing agents (for example, benzalkoniumchloride and procaine hydrochloride), stabilizing agents (for example,human serum albumin and polyethylene glycol), preservatives (forexample, benzyl alcohol and phenol), antioxidants, etc. The injectionsthus prepared are usually filled into appropriate ampuls. Although thedosage varies depending upon the symptom, the oral dosage is generallyabout 0.1 to 100 mg per day, preferably 1.0 to 50 mg, and morepreferably 1.0 to 20 mg, for adults (taken as 60 kg). When thepreparations are parenterally given, the dosage varies depending uponthe object to which the preparations are given, the organ to which theyare given, the symptom, the route of administration, etc. For example,when the preparations are given in the injection form, it isadvantageous that they are intravenously injected in a dosage of about0.01 to 30 mg per day, preferably 0.1 to 20 mg, and more preferably 0.1to 10 mg, for adults (taken as 60 kg).

(3) Example 1 below and FIG. 31 have proved that the PACAP receptorproteins of the present invention bind to the PACAPs. Further, Examples5, 7 and 8 have revealed that the DNAs coding for bovine, rat and humanPACAP receptor proteins can be expressed on cell membranes, and thePACAP receptor proteins expressed can react with the PACAPs to increasethe amount of cyclic AMP and (or) the concentration of inositolphosphate in cells. Further Example 11 has revealed that compoundsinhibiting the binding of PACAPs to PACAP receptors can be screened byusing the membrane fractions of the Sf9 cells in which the human PACAPreceptor is expressed by use of Baculoviridae. Accordingly, the presentinvention gives a method for determining

(i) an effect of a test compound on PACAP receptor activity comprisingcomparing PACAP receptor activities in cases of (a) and (b);

(a) contacting PACAP receptor with a PACAP;

(b) contacting PACAP receptor with a PACAP and a test compound, or

(ii) an effect of a test compound on binding of PACAP to PACAP receptorcomprising comparing an amount of binding of PACAP to PACAP receptor incases of (a) and (b);

(a) contacting PACAP receptor with a PACAP;

(b) contacting PACAP receptor with a PACAP and a test compound.

The present invention further gives an assay for quantifying a testcompound's effect

(i) on PACAP receptor activity comprising comparing an amount of PACAPreceptor activation in cases of (a) and (b);

(a) contacting PACAP receptor with a PACAP;

(b) contacting PACAP receptor with a PACAP and a test compound, or

(ii) on binding of PACAP to PACAP receptor comprising comparing anamount of binding of PACAP to PACAP receptor in cases of (a) and (b);

(a) contacting PACAP receptor with a PACAP;

(b) contacting PACAP receptor with a PACAP and a test compound.

As the PACAP receptor in the above screening method, the PACAP receptorof the present invention, the receptor fragment of the present inventionor the PACAP receptor produced by cultivating a transformant containingthe DNA encoding the PACAP receptor of the present invention.

Compounds or their salts obtained by the above screening method includecompounds activating PACAP receptor or compounds antagonizing binding ofa PACAP to a PACAP receptor.

As the above mentioned, compounds activating PACAP receptors (forexample, peptides, proteins and natural or nonnatural compounds),namely, PACAP receptor antagonists, or compounds antagonisticallyinhibiting the binding of PACAPs to receptors (for example, peptides,proteins and natural or nonnatural compounds), namely PACAP receptorantagonists, can be screened by using the PACAP receptor proteins of thepresent invention, the partial peptides thereof or the salts thereof, orby the PACAP receptor proteins which are obtained by cultivatingtransformants containing a DNA encoding PACAP receptor protein. ThesePACAP receptor agonists or PACAP receptor antagonists can be furthertested for use as drugs useful in protection of nerve cells and growthmaintenance of nerve cells in vivo, for example, therapeutic compositionfor neuropathy such as Alzheimer's disease.

Until human PACAP receptor of the present invention was found, forexample, when substances which inhibit a binding of PACAP to human PACAPreceptor were screened, the following steps were necessary:

Obtaining a PACAP receptor of other than human such as bovine orporcine; screening substances which inhibit a binding of the bovine orporcine PACAP receptor and PACAP; and checking whether the pickedsubstances have real affinity on human PACAP receptor.

Meanwhile, human PACAP receptor makes the screening of substances whichinhibit binding of human PACAP receptor and PACAP easy and effective.The thus obtained PACAP receptor agonists or PACAP receptor antagonistsmay be further tested drugs useful for protecting nerve cells ormaintaining growth of nerve cells in vivo such as for therapeuticcomposition for nervous diseases such as Alzheimer's disease or formaintaining growth of nerve cell in vitro.

The screening methods of the present invention will be described belowin detail.

(I) Methods for Screening Compounds Antagonistically Inhibiting theBinding of PACAPs to PACAP Receptors

PACAP receptor proteins used for screening are preferably membranefractions of organs of warm-blooded animals. For example, human PACAPreceptor protein expressed in large amounts by use of recombinants issuitable, because it is very difficult to obtain human-derived organs.

The above-mentioned methods are used for the production of the PACAPreceptor proteins, and performed by expressing DNAs coding for saidproteins in animal cells (for example, human cells) or insect cells. Inparticular, they are preferably expressed in the insect cells.

Complementary DNAs are used as the DNA fragments coding desiredportions, but the DNA fragments are not necessarily limited thereto. Forexample, gene fragments or synthetic DNAs may be used. In order tointroduce the DNA fragments coding for the PACAP receptor proteins intohost cells and express them efficiently, it is preferred that said DNAfragments are ligated downstream from polyhedrin promoters of insectnuclear polyhedrosis viruses (NPVs) belonging to Baculoviridae. Vectorsinclude two viruses of Autograph california NPV (ACNPV) belonging toKinuwaba and Bombyx mori NPV (BmNPV) of silk worms. Baculoviridae hascyclic double stranded DNA (130 kb), and shows no infectivity to spinalanimals and plants at all. Virus DNA is so long as 130 kb, so that it isdifficult to directly insert the DNA fragment wanted to be expresseddownstream from the polyhedrin promoter. Then, actually, a polyhedringene portion containing a promoter portion is cut out from a virus, andincorporated in an E. coli vector such as pUC18 to prepare a transfervector. Subsequently, a desired DNA fragment is inserted downstream froma polyhedrin promoter of the transfer vector, and an insect cell isconcurrently infected therewith, together with baculovirus DNA, followedby cultivation. Homologous recombination is allowed to take place in theinsect cell to obtain a recombinant baculovirus. The recombinant virusforms the desired product freshly introduced, instead of forming apolyhedrin. When the virus is AcNPV, a yatoga caterpillar-derivedestablished cell line (Spodoptera frugiperda cell; Sf cell) is used as ahost cell. When the virus is BmNPV, a silk worm-derived established cellline (Bombyx mori N; BmN cell) is used. As expression systems usingbaculoviruses and insect cells, commercial systems can be employed (forexample, MAXBAC™, Invitrogen), and procedures described in theexperimental descriptions attached thereto and in Bio/Technology, 6,47-55 (1988) can also be adopted. The amount and quality of theexpressed receptor can be examined by methods known per se in the art,for example, the method described in P. Nambi et al. J. Biol. Chem.,267, 19555-19559 (1992).

In the screening methods of the present invention, as the PACAP receptorproteins, either cells containing the proteins or proteins fractions ofcells containing the proteins may be used. Further, membrane fractionsof insect cells containing the proteins are most preferably used.

Said cells means host cells in which the PACAP receptor proteins areexpressed. Said host cells include E. coli, Bacillus subtilis, yeast,insect cells and animal cells (for example, human cells), and the insectcells are preferred among others.

The membrane fractions means fractions in which cell membranes obtainedby methods known per se in the art after cell disruption are containedin large amounts. The disruption of the cells is carried out preferablyat 0° to 4° C., and physiological saline or a buffer such as 50 mMTris-HCl is used. A protease inhibitor is preferably added to preventdecomposition of the protein. Methods for disrupting the cells includethe method of crushing the cells with a Potter-Elvehjem typehomogenizer, disruption with a Working blender or a Polytron homogenizer(Kinematica), disruption by ultrasonication, and disruption by allowingthe cells to jet through a fine nozzle under pressing with a Frenchpress, etc. Fractionating methods utilizing centrifugal force such asdifferential centrifugation and density gradient centrifugation aremainly used for fractionation of the cell membranes. For example, a celldisrupted solution is centrifuged at a low speed (500 to 3000 rpm) for ashort period of time (usually about 1 to 10 minutes), and thesupernatant is further centrifuged at a high speed (15000 to 30000 rpm),usually for 30 minutes to 2 hours. The resulting precipitate is taken asthe membrane fraction. In said membrane fraction, the expressed PACAPreceptor protein and membrane compositions such as cell-derivedphospholipids and membrane proteins are contained in large amounts.

The amount of the PACAP receptor proteins in the cells or the membranefractions containing the PACAP receptor proteins is preferably 10³ to10⁸ molecules per cell, and suitably 10⁵ to 10⁷ molecules per cell. Amore expression amount results in higher PACAP binding activity permembrane fraction (specific activity). Not only construction of a highsensitive screening system becomes possible, but also a large amount ofsamples can be measured in the same lot.

In order to screen compounds antagonistically inhibiting the binding ofa PACAP to a PACAP receptor, an appropriate PACAP receptor fraction anda labeled PACAP (for example, PACAP27 or PACAP38, hereinafter referredto as a PACAP) are required. Desirable examples of the PACAP receptorfractions include natural PACAP receptor proteins and recombinant PACAPreceptor proteins equivalent thereto. As the labeled PACAPs, PACAP27labeled with ¹²⁵ I! (du Pont), etc. are commercially available. They cantherefore be utilized.

When the compounds antagonistically inhibiting the binding of the PACAPto the PACAP receptor is screened, cells or cell membrane fractionscontaining the PACAP receptor protein are first suspended in a buffersuitable for screening, thereby preparing a receptor sample. The buffermay be any, as long as it is a buffer which does not inhibit the bindingof the PACAP to the receptor, such as phosphate buffer or Tris-HClbuffer having a pH of 4 to 10 (preferably a pH of 6 to 8). For thepurpose of decreasing non-specific binding, a surface active agent suchas CHAPS, Tween-80™ (Kao-Atlas), digitonin or deoxycholate may also beadded to the buffer. Further, for the purpose of inhibitingdecomposition of the receptor or a ligand with a protease, a proteaseinhibitor such as PMSF, leupeptin, E-67 (Peptide Laboratory) orpepstatin can also be added. A definite amount (5000 to 500000 cpm) of¹²⁵ I!PACAP is added to 0.01 to 10 ml of the receptor solution, and 10⁻⁴to 10⁻¹⁰ M specimen compound, fermentation products, etc. are allowed tocoexist at the same time. In order to know the non-specific binding(NSB), a reaction tube to which a ligand is added in large excess isprepared. Reaction is conducted at 0° to 50°C., desirably at 4° to 37°C. for 20 minutes to 24 hours, desirably for 30 minutes to 3 hours.After reaction, the reaction product is filtered through a glass fiberfilter and washed with an appropriate amount of the same buffer,followed by measurement of ¹²⁵ I! remaining on the glass fiber filterwith a γ-counter. When the count (B₀ -NSB) obtained by subtracting NSBfrom the count (B₀) in the absence of an antagonistic substance is takenas 100%, the specimen compound, the fermentation products, etc giving anon-specific binding (B-NSB) of 50% or less can be selected as potentialmaterials having antagonistic ability.

Examples of kits for screening the compounds antagonistically inhibitingthe binding of the PACAPs to the PACAP receptors of the presentinvention include the following:

1. Reagents for Screening

    ______________________________________    (A) Buffer for Measurement    ______________________________________    Tris-HCl               2.4    g    Magnesium acetate.4H.sub.2 O                           1.07   g    EGTA                   0.76   g    NaN.sub.3              0.6    g    Leupeptin              20     mg    E-64                   4      mg    ______________________________________

These are dissolved in 997 ml of distilled water.

    ______________________________________           Pepstatin     1      mg           PMSF          0.09   g    ______________________________________

These are dissolved in 1 ml of DMSO, and the resulting solution is addedto 997 ml of the above-mentioned water. About 2 ml of 6N HCl is addedthereto to adjust to pH 7.2. One gram of BSA is dissolved therein,followed by storage at 4° C.

(B) Buffer for Washing

    ______________________________________            CHAPS 0.45 g    ______________________________________

This is dissolved in 900 ml of the buffer for measurement and thesolution is stored at 4° C.

(C) PACAP Receptor Sample

A membrane fraction of insect cells (Sf9) in which a PACAP receptorprotein is expressed is diluted with the buffer for measurement to 0.5to 5 μg of protein/ml before use.

(D) ¹²⁵ I! Labeled PACAP

    ______________________________________    (3- .sup.125 I!iodotyrosyl)    PACAP (du Pont)  185 kBg    ______________________________________

Fifty microliters of distilled water is added thereto to dissolve it,and 450 μl of the buffer for measurement is added thereto. The solutionis stored at -20° C.

(E) PACAP Standard Solution

The PACAP (Peptide Laboratory) is diluted with 50% DMSO to 10⁻⁴ M, andstored at -20° C. This is diluted 10 times with the buffer formeasurement before use.

2. Assays

(i) The membrane fraction of Sf9 cells containing the PACAP receptorprotein J. L. Vaughn et al., In Vitro, 13, 213-217 (1977)! is dilutedwith the buffer for measurement to give 1 μg of protein/ml, and 100 μlthereof is poured into each tube (Falcon).

(ii) After addition of 3 μl of 10⁻⁴ to 10⁻¹⁰ M specimen or 10 μl or lessof fermentation products, 2 μl of ¹²⁵ I! labeled PACAP is added,followed by reaction at 25° C. for 60 minutes. In order to examine thenon-specific bonding, 3 μl of 10⁻⁵ M PACAP is added instead of thespecimen.

(iii) The buffer for washing (1.5 ml) is added, and filtration isconducted through a glass fiber filter (GF/F, Whatman). Then, 1.5 ml ofthe same buffer is further added to the residue in the tube, andfiltration is conducted again.

(iv) ¹²⁵ I! remaining on the glass fiber filter is measured with aγ-counter, and the percent maximum binding (PMB) is determined from thefollowing equation;

    PMB= (B-NBS)/(B.sub.0 -NBS)!×100

PMB: percent maximum binding

B: value when the specimen is added

NBS: non-specific binding

B₀ : maximum binding

(II) Methods for Screening Compounds Activating the PACAP Receptors

The compounds antagonistically inhibiting the binding of the PACAPs tothe PACAP receptor proteins selected by the methods of (I) describedabove is expected to contain compounds activating the PACAP receptorproteins similarly to the PACAPs (compounds having PACAP receptoragonist activity). Such compounds can be evaluated by secondaryscreening systems based on acceleration of cyclic AMP production asdescribed below.

First, cells in which the PACAP receptor protein is expressed aresubcultured to a 48-well plate for tissue culture in a ratio of 1×10⁵cells/well, and cultured for 2 days. Then, the medium is removed, andthe plate is washed twice with serum-free medium. Subsequently, 300 μlof the same medium is added to each well as a reaction solution. Theserum-free medium may be any, as long as it is a medium for cellculture, and bovine serum albumin, etc. may be added for the purpose ofpreventing the compounds added from being non-specifically adsorbed bythe instruments, etc. Further, for the purpose of inhibitingdecomposition of cyclic AMP produced to enhance assay sensitivity,addition of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesteraseinhibitor, is effective. The specimen compound having a finalconcentration of 10⁻⁴ to 10⁻¹⁰ M and fermentation products are added toeach well. In order to know non-specific response, wells containing onlythe solvent in which the compounds are dissolved are prepared. Reactionis usually conducted at 4° to 42° C. for 10 minutes to 2 hours,preferably at room temperature to 37° C. for 20 minutes to 1 hour. Afterreaction, the supernatant is removed by suction. After washing with twoportions of the reaction solution, cyclic AMP produced is extracted with200 μl of 100% ethanol. Ethanol is removed with a centrifugal freezedryer, and the residue is dissolved in 100 μl of a buffer fordetermination of cyclic AMP. Reagents for determination of cyclic AMP,including the buffer, may be ones commercially available as kitsaccording to either radio immunoassay (RIA) or enzyme immunoassay (EIA)(Amersham, du Pont, etc.). When the production amount of cyclic AMPwhich has become clear by determination is statistically significantlyhigh, compared with the case where the sample is not added or the casewhere only the solvent in which the sample is dissolved is added, suchcompound can be selected as potential compounds having PACAP receptoragonist activity. In order to eliminate the probability that the cyclicAMP production promoting action of the potential compounds isnon-specific action to cells or action through receptors other than thePACAP receptor, it is necessary to confirm that the potential compoundsexhibit no cyclic AMP production promoting action in cells in which thePACAP receptor protein is not allowed to be expressed. As an indicationfor PACAP receptor agonist activity, production promotion of inositoltriphosphate or diacylglycerol and an increase in intracellular calciumconcentration, as well as the production promotion of cyclic AMP, may beemployed. However, the production promotion of cyclic AMP is superiorfrom the viewpoint of treating the sample in large amounts. Suchscreening methods of the present invention are excellent methods bywhich compounds having action similar to that of the PACAP or higherthan the PACAP and excellent in resistance against proteases, comparedwith the PACAP, a peptide, can be selected.

Antibodies or antiserum to the PACAP receptor proteins of the presentinvention, the partial peptides thereof or the salts thereof may be anyantibodies or antiserum as long as they can recognize the PACAP receptorproteins, the partial peptides thereof or the salts thereof. Forexample, monoclonal antibodies such as PRN1-25a, PRN1-109a and PRN1-159aagainst a partial peptide (MHSDAIFKKEQAMC) are preferable. The partialpeptide was prepared by substituting the 5th Cys(C) of a partial peptidewhich has a partial amino acid sequence (1st to 14th amino acid sequenceof SEQ ID NO:14) common to bovine, rat or human PACAP receptor which hasamino acid sequence of anyone of SEQ ID NO:14 to SEQ ID NO:29 to Ala(A),for the convenience of preparation of immunoantigen complexes.

Antibodies or antiserum to the PACAP receptor proteins of the presentinvention, the partial peptides thereof or the salts thereof can beproduced by methods known per se in the art, using the PACAP receptorproteins, the partial peptides thereof or the salts thereof as antigens.The antibodies or antiserum thus obtained can be used for quantitativeanalysis or detection of the PACAP receptor proteins of the presentinvention, the peptides thereof or the salts thereof, more detailedutilities are as follows:

(1) By using the antibodies or antiserum for Western blotting or immuneprecipitation, the PACAP receptor proteins, the partial peptides thereofor the salts thereof can be detected.

(2) An affinity column to which the antibodies of the present inventionare fixed, can purify the PACAP receptor proteins, the partial peptidesthereof or the salts thereof.

(3) The antibodies of the present invention can be used as a PACAPreceptor antagonist, as shown in Example 12, since the antibodies blockPACAP action by inhibiting binding of PACAP and a PACAP receptor.

As a signal peptide of the PACAP receptor protein of the presentinvention, for example, a peptide which has 1st to 37th amino acidsequence of SEQ ID NO:15, a peptide which has 1st to 37th amino acidsequence of SEQ ID NO:17, a peptide which has 1st to 19th amino acidsequence of SEQ ID NO:19, a peptide which has 1st to 19th amino acidsequence of SEQ ID NO:21, a peptide which has 1st to 77th amino acidsequence of SEQ ID NO:23, a peptide which has 1st to 77th amino acidsequence of SEQ ID NO:25, a peptide which has 1st to 77th amino acidsequence of SEQ ID NO:27, a peptide which has 1st to 77th amino acidsequence of SEQ ID NO:29, a peptide which has 58th to 77th amino acidsequence of SEQ ID NO:23, a peptide which has 58th to 77th amino acidsequence of SEQ ID NO:25, a peptide which has 58th to 77th amino acidsequence of SEQ ID NO:27 or a peptide which has 58th to 77th amino acidsequence of SEQ ID NO:29 may be used. These signal peptides can besynthesized by conventional methods such as a peptide synthesizer orprepared by cutting the amino acid bond of the PACAP receptor of thepresent invention with an enzyme.

The salts of the signal peptides of the present invention includesimilar salts as those for PACAP receptors or partial peptides thereof.

A DNA which encodes a signal peptide may be any one which has anucleotide sequence encoding the signal peptide and includes a DNA whichhas 1st to 111th nucleotide sequence of SEQ ID NO:30, a DNA which has1st to 111th nucleotide sequence of SEQ ID NO:31, a DNA which has 1st to57th nucleotide sequence of SEQ ID NO:32, a DNA which has 1st to 57thnucleotide sequence of SEQ ID NO:33, a DNA which has 1st to 231stnucleotide sequence of SEQ ID NO:34, a DNA which has 1st to 231stnucleotide sequence of SEQ ID NO:35, a DNA which has 1st to 231stnucleotide sequence of SEQ ID NO:36, a DNA which has 1st to 231stnucleotide sequence of SEQ ID No:37, a DNA which has 172nd to 231stnucleotide sequence of SEQ ID NO:34, a DNA which has 172nd to 231stnucleotide sequence of SEQ ID NO:35, a DNA which has 172nd to 231stnucleotide sequence of SEQ ID NO:36, a DNA which has 172nd to 231stnucleotide sequence of SEQ ID NO:37 or a DNA which comprises one ofthese DNAs. These DNAs encoding signal peptides of the present inventioncan be synthesized by conventional method such as a peptide synthesizeror prepared by cutting the DNA (cDNA is preferable) which encodes thePACAP receptor of the present invention with an appropriate restrictiveenzyme.

The DNA coding for the signal peptide of the PACAP receptor proteins ofthe present invention may stimulate an expression of a membrane-boundpeptide such as a receptor into a membrane. For example, a protein whichdoes not or rately expresses into a membrane can be expressed on themembrane effectively by linking a DNA coding for a signal peptide of thePACAP receptor proteins of the present invention upstream from the DNAwhich rarely or does not express the desired protein on the membrane inan expression.

The present invention will be described in more detail through thefollowing examples. It is understood of course that they are notintended to limit the scope of the invention.

Transformant E. coli pBPR-T containing pBPRT and transformant E. colipBPR114 containing pBPR114 each obtained in Example 2 given later weredeposited with the National Institute of Bioscience and Human-technology(NIBH), the Agency of Industrial Science and Technology, the Ministry ofInternational Trade and Industry, Japan, under the accession numbersFERM BP-4338 and FERM BP-4339, respectively, on Jun. 15, 1993, anddeposited with Institute for Fermentation, Osaka, Japan (IFO) under theaccession numbers IFO 15572 and IFO 15571, respectively, on Nov. 5,1993.

Transformant E. coli pRPACAPR 12 containing pRPACAPR 12 and transformantE. coli pRPACAPR 46-5 containing pRPACAPR each obtained in Example 3given later were deposited with NIBH, under the accession numbers FERMBP-4254 and FERM BP-4255, respectively, on Apr. 5, 1993, and depositedwith IFO under the accession numbers IFO 15469 and IFO 15470,respectively, on Apr. 15, 1993.

Transformant E. coli MV1184/pTS847-1 containing pTS847-1 obtained inExample 4 given below was deposited with the NIBH under the accessionnumber FERM BP-4280, and deposited with IFO under the accession numberIFO 15570 on Nov. 5, 1993.

Transformant E. coli pHPR15A containing pHPR15A obtained in Example 4given below; Transformant E. coli pHPR55A containing pHPR55A andTransformant E. coli pHPR66P containing pHPR66P were deposited with theNIBH under the accession number FERM BP-451₁, FERM BP-4510 and FERMBP-4509, respectively on Dec. 22, 1993, and deposited with IFO under theaccession number IFO 15603, 15604 and 15605, respectively on Dec. 20,1993.

Hybridoma PRN1-159 obtained in Example 12 given below was deposited withNIBH under the accession number FERM BP-4554 on Feb. 8, 1994, anddeposited with IFO under the accession number IFO 50427 on Feb. 8, 1994.

EXAMPLES Example 1! Production (Purification) of Bovine-Derived PACAPReceptor Protein

The following procedure was conducted in a low temperature laboratory at4° C.

(1) Preparation of Membrane Fractions

Membrane fractions were prepared from the bovine cerebrums according toa method in which the known method described in Biochem. Biophys. Res.Commun., 172, 709-714 (1990) was partially modified. The fresh bovinecerebrums (1.5 kg) were homogenized 3 times in 6 liters of buffer A (20mM Tris, 10 mM EDTA, 0.25M sucrose, 0.5 mM PMSF, 20 μg/ml leupeptin, 4μg/ml E-64 and 1 μg/ml pepstatin, pH 7.4) with a Polytron homogenizer(Kinematica) for 30 seconds. The resulting homogenate was centrifugedwith a high speed cooling centrifuge CR26H, Roter RR10A (Hitachi, Ltd.)at 680×g for 20 minutes to obtain a supernatant. The resultingsupernatant was further ultracentrifuged with an ultracentrifuge SCP70H,Roter RPZ35T (Hitachi, Ltd.) at 100,000×g for 60 minutes to obtainpellets. The pellets were suspended in 400 ml of buffer B (20 mM Tris, 5mM EDTA, 0.5 mM PMSF, 20 μg/ml leupeptin, 4 μg/ml E-64 and 1 μg/mlpepstatin, pH 7.4) to prepare a membrane fraction suspension.

(2) Solubilization of the PACAP Receptor Protein from the MembraneFractions

The membrane fraction suspension obtained in (1) described above (400ml) was diluted with 5 liters of buffer B to give a membrane proteinconcentration of 1 mg/ml, and digitonin was added thereto to provide aconcentration of 1%. The resulting suspension was slowly stirred for 1hour, and then, ultracentrifuged with an ultracentrifuge SCP70H, RoterRPZ35T (Hitachi, Ltd.) at 100,000×g for 1 hour to obtain a supernatant.The resulting supernatant was used as a solubilized membrane proteinfraction.

(3) Assay of Receptor Activity of the PACAP Receptor Protein

PACAP receptor activity was assayed according to the saturation bindingexperiment method using ¹²⁵ I!PACAP27 and the antagonistic bindingexperiment method Biochem. Biophys. Res. Commun., 171, 838-844 (1990)and Biochem. Biophys. Res. Commun., 172, 709-714 (1990)!. The testsample (membrane fraction or solubilized membrane protein fraction) wasappropriately diluted with buffer D (20 mM Tris, 5 mM magnesiumchloride, 0.1% BSA and 0.05% digitonin, pH 7.4). In the saturationbinding experiment, 0.1 ml of the diluted test sample was mixed with 10μl of ¹²⁵ I!PACAP27 (final concentration: 20 to 50 pM), and reacted at25° C. for 1 hour. In the competitive binding experiment, the dilutedtest sample was mixed with 2 μl of ¹²⁵ I!PACAP27 (final concentration:100 pM) and 3 μl of an unlabeled peptide (PACAP27, PACAP38 or VIP)having a variable concentration, and reacted in a similar manner. To 0.1ml of this reaction solution, 1.5 ml of buffer E (0.1% BSA, 0.05% CHAPS,20 mM Tris and 5 mM magnesium chloride, pH 7.4) cooled with ice wasadded, and immediately, the mixed solution is filtered through a glassfiber filter. The glass fiber filter used had previously been treatedwith 0.3% polyethylene imine. The radioactivity of the filter wascounted with a γ-ray counter, thereby determining ¹²⁵ I!PACAP27 bound tothe receptor. In order to determine the non-specific binding, theabove-mentioned experiment was carried out in the presence of 1 μMPACAP27. The specific binding was calculated by subtracting thenon-specific binding from the total binding measured in the absence ofPACAP27. Results of the saturation binding experiment were subjected toScatchard plot analysis to determine the dissociation constant and themaximum binding.

(4) Crude Purification of the PACAP Receptor Protein

A method for purifying the PACAP receptor from the solubilized membraneprotein fraction by ion exchange chromatography and hydroxyapatitechromatography is described below.

The solubilized membrane protein fraction 2400 mg (4800 ml)! was loadedonto an ion exchange column (for example, anion exchange chromatographysuch as DEAE-TOYOPEARL) equilibrated with 1 liter of buffer C, at a flowrate of 9 ml/minute. Then, the concentration of sodium chloride inbuffer C (20 mM Tris, 1 mM EDTA, 0.5 mM PMSF, 20 μg/ml leupeptin, 4μg/ml E-64 and 1 μg/ml pepstatin, pH 7.4) supplemented with 0.1%digitonin was gradually increased from 0M to 1M for 170 minutes to elutethe PACAP receptor from the column. The PACAP receptor activity of eacheluted fraction was assayed by the above-mentioned method. The activefractions eluted from the ion exchange column were further loaded onto ahydroxyapatite column (HCA-100, 5 cm in diameter and 7 cm in length) ata flow rate of 7 ml/minute. This column was washed with 500 ml of 0.1Mphosphate buffer containing 0.1% digitonin, and then, the PACAP receptorwas eluted with 500 ml of 0.6M phosphate buffer containing 0.1%digitonin at a flow rate of 7 ml/minute. The active fractions wereconcentrated 10-fold using an ultrafilter, and further desalted byrepetition of dilution and concentration with a 6-fold excess of bufferC in relation to the volume of the concentrated sample.

(5) Purification of the PACAP Receptor by Affinity Chromatography

(5-1) Preparation of Affinity Ligand

A method for preparing a biotinated PACAP used in affinitychromatography is described below. One equivalent of the PACAP27derivative (having cysteine as the 28th amino acid residue, PACAP27-Cys)synthesized by the solid phase method was dissolved in 50 mM phosphatebuffer (pH 7.0) supplemented with 3 mM EDTA and 0.5M NaCl to provide aconcentration of 2×10⁻⁴, and a 10 mM biotinylating reagent (biotin-HSDP)dissolved in DMF was added thereto to give 10 equivalents, followed byreaction overnight. The reaction product, biotinylated PACAP27(PACAP27-Cys-biotin) represented by the following formula, was purifiedon a reverse phase HPL chromatography: ##STR16##

Namely, the reaction product was loaded onto a reverse phase column (ODS80-TM, Tosoh) equilibrated with 60 ml of distilled water containing0.05% TFA, and the concentration of acetonitrile was gradually increasedfrom 20% to 40% for 60 minutes at a flow rate of 1 ml/minute at roomtemperature to conduct separation. Peak fractions of biotinylatedPACAP27 were fractionated, and chromatographed again under the sameconditions (FIG. 28) to obtain pure biotinylated PACAP27, followed bylyophilization. It was confirmed by the competitive binding experimentthat biotinylated PACAP27 has an affinity similar to that of PACAP (FIG.29).

(5-2) Affinity Chromatography

Avidin-agarose was suspended in a solution containing the PACAP receptorprotein crudely purified by the method described above, and gentlystirred overnight. Avidin-agarose was removed by filtration to obtain afiltrate. About 20-fold equivalents of biotinated PACAP27 in relation tothe amount of the receptor was added to this filtrate, and allowed toreact overnight. Further, 80 ml of avidin-agarose was suspended therein,and gently stirred for 4 days. This avidin-agarose was packed into acolumn, and washed with 500 ml of buffer C containing 1M sodium chlorideand 0.1% digitonin at a flow rate of 1.5 ml/minute, followed by elutionof the PACAP receptor protein with 180 ml of a buffer (20 mM magnesiumacetate 1M sodium chloride and 10% glycerol, pH 4.0) at a flow rate of1.5 ml/minute. The eluate was immediately neutralized with 1/4 volume of1M Tris (pH 7.5) with respect to the eluate.

(6) Final Purification after Affinity Chromatography

The PACAP receptor protein purified by the above-mentioned affinitychromatography was loaded onto a microcolumn (1.8 ml) of hydroxyapatiteat a flow rate of 0.3 ml/minute, and washed with 20 ml of 0.1M phosphatebuffer containing 0.1% digitonin at a flow rate of 0.3 ml/minute,followed by elution of the PACAP receptor from the column with 20 ml of0.6M phosphate buffer containing 0.1% digitonin at a flow rate of 0.3ml/minute. The active fractions eluted were concentrated using anultrafilter (CENTRICON 10™, Amicon). The active fractions concentratedwere gel filtered on a gel filtration column (for example, Superrose 6Column, Pharmacia) equilibrated with 60 ml of buffer C containing 0.1%digitonin and 0.2M NaCl at a flow rate of 0.4 ml/minute. The activefractions eluted were used as a purified PACAP receptor protein sample.

One embodiment of the purification procedure conducted by theabove-mentioned methods is summarized in Table 2.

The specific activity (mole number of PACAP binding to unit weight ofprotein) of the final purified sample determined by the saturationbinding experiment using ¹²⁵ I!PACAP27 was usually 15,000 pmoles/mg ofprotein or more. Further, the calculation of the dissociation constantfrom results of the saturation binding experiment revealed that thedissociation constant of the final purified sample approximately agreeswith that of the PACAP receptor existing in the membrane fractions, andthat the purified PACAP receptor protein has a sufficiently highaffinity for the PACAP (FIG. 30). Furthermore, results of thecompetitive binding experiment for the purified PACAP receptor proteinproved that it has the property of reacting with PACAP27 and PACAP38,but not reacting with VIP (FIG. 31). Analysis results obtained bypolyacrylamide electrophoresis for the final purified sample in thepresence of sodium dodecylsulfate are shown in FIG. 28. The resultsindicate that the final purified sample is composed of a substantiallypure protein (molecular weight: about 57,000). This protein having amolecular weight of about 57,000 is the PACAP receptor protein occurringin the bovine cerebrums.

                  TABLE 2    ______________________________________             Total Total   Specific  Purifi-                                           Activity             activity                   protein activity  cation                                           yield             (pmole)                   (mg)    (pmole/mg)                                     (fold)                                           (%)    ______________________________________    Membrane fraction               8115    6400    1.3    Solubilized product               4561    2400    1.9     1     100    Ion exchange               4700     475    9.9     5.2   103    Hydroxyapatite               3349     134    25.0    13.2  73    Avidin-agarose               2046    ND      ND            45    Micro hydroxy-               1717    ND      ND            38    apatite    Gel filtration                671    0.042   16000   8400  15    ______________________________________     Total activity: The maximum binding of  .sup.125 I!PACAP27 obtained by th     saturation binding experiment     ND: Not determined

Example 2! Screening of Bovine PACAP Receptor Protein cDNA and DNASequence Analysis

(1) Preparation of Bovine Hippocampus Poly(A)⁺ RNA Fractions andConstruction of cDNA Library Using the Same

Total RNA fractions were prepared from the bovine hippocampi accordingto the guanidine-fluoroacetate method Method in Enzymology, 154, 3(1987) and Biochemistry, 18, 5294 (1978)!, and poly(A)⁺ RNA fractionswere further separated by the use of an oligo(dT) cellulose-spun-column(Pharmacia). Using these fractions as a starting material, a bovinehippocampus cDNA library in which a vector was λgt11 was constructed bythe use of a cDNA cloning kit (Amersham). The library prepared had about4×10⁶ pfu (plaque forming unit) of independent clones.

(2) Preparation of Probe

A synthetic DNA was prepared as a probe, based on the N-terminal aminoacid sequence having the amino acid sequence represented by SEQ ID NO:38 of the bovine PACAP receptor protein obtained in Example 1. Sequence:##STR17##

(3) Screening

The λgt11 phage cDNA library (bovine brain, Clontech) (1.5×10⁶ pfu)prepared in Example 2 (1) was mixed with magnesium sulfate-treated E.coli Y1090, and incubated at 37° C. for 15 minutes. Then, 0.5%agarose/LB was added thereto, followed by plating on a 1.5% agar/LBplate. A nitrocellulose filter was placed on the plate on which a plaqueis formed, and the plaque transferred onto the filter. After alkalitreatment of this filter, the DNA was fixed by heating at 80° C. for 3hours. This filter was hybridized with the labeled probe in ahybridization buffer 0.5M phosphate buffer (pH 7.2), 1% bovine serumalbumin, 7% SDS and 1 mM EDTA! overnight at 50° C. The labeling of theprobe was conducted according to the method of phosphorylation of the5'-terminus of the probe with γ-³² P!ATP and T4 polynucleotide kinase(Nippon Gene). Washing was carried out with 2×SSC, 0.1% SDS at 48° C.for 1 hour, and then, hybridized clones were detected by autoradiographyat -80° C. As a result, a cDNA encoding a N-terminal portion of thePACAP receptor was obtained, and the cDNA designated as λBPR35.

Further, the bovine brain-derived cDNA library (Clontech) (1.5×10⁶ pfu).was screened, using the cDNA portion of λBPR35 as a probe, to obtain acDNA encoding C-terminal portion of the PACAP receptor. At this time, abuffer was used which comprised 5×Denhardt's solution 0.02% bovine serumalbumin (Sigma)!, 5×SSPE (0.15M sodium chloride, 0.01M monosodiumphosphate and 1 mM EDTA), 0.1% SDS and 100 μg/ml of heat-denaturedsalmon sperm DNA (Sigma), and incubation was conducted overnight at 65°C. together with the labeled probe to hybridize. The labeling of theprobe was carried out by the use of a multi-prime DNA labeling kit(Amersham). Washing was carried out with 0.2×SSC, 0.1% SDS at 60° C. for1 hour, and then, hybridized clones were detected by autoradiography at-80° C. A cDNA clone which encoded a portion of the PACAP receptor wasobtained, and the cDNA was designated as λBPR114. Using the cDNA portionof the resulting pBPR114 as a probe, the cDNA library (4×10⁻⁶ pfu)prepared from the bovine hippocampus poly(A)⁺ RNA fractions was screenedto obtain a cDNA encoding the C-terminal portion of the PACAP receptor.The conditions at this time were the same as those at the time when theabove-mentioned λBPR114 was screened. As a result, a cDNA clone encodinga C-terminal portion of the PACAP receptor was obtained, and the cDNAwas designated as λBPR68.

(4) Subcloning of cDNA Clones and DNA Sequence Analysis

An inserting portion of the resulting cDNA clone was cut out by cleavagewith EcoRI, and subcloned into plasmid vector pUC118 to obtain pBPR35,pBPR114 or pBPR68. The plasmid was further cleaved stepwise byexonuclease digestion, or self cyclized or subcloned after cleavage withan appropriate restriction enzyme (NcoI, BamHI, etc.) to prepare atemplate DNA for sequence analysis. For sequence determination, thedideoxy chain termination method using RI marker dCTP and a fluorescentDNA sequencer (Applied Biosystems) were used, and for data analysis, aDNASIS (Hitachi Software Engineering) was used. Further, pBPR35 andpBPR68 were recombined at the BamHI sites to prepare pBPR-T. The BamHIand AvaII fragments of pBPR114 having disappeared regions can berecombined with pBPR-T by the use of known genetic engineeringtechnique, thereby preparing PACAP receptor cDNA (pBPR-TD) containing noinsertion.

Results of analysis revealed that pBPR-T has the nucleotide sequence ofSEQ ID NO: 38, and that pBPR-TD has the nucleotide sequence of SEQ IDNO: 39.

Example 3! Screening of Rat PACAP Receptor Protein cDNA and DNA SequenceAnalysis

(1) Preparation of Rat Brain Poly(A)⁺ RNA Fractions and Construction ofcDNA Library Using the Same

Total RNA fractions were prepared from the rat brains according to theguanidine-isothiocyanate method Biochemistry, 18, 5294 (1979)!, andpoly(A)⁺ RNA fractions were further separated by the use of an oligo(dT)cellulose-spun-column (Pharmacia). Using these-fractions as a startingmaterial, a rat brain cDNA library in which a vector was λgt11 wasconstructed by the use of a cDNA cloning kit (Amersham). The libraryprepared had about 3×10⁶ pfu (plaque forming unit) of independentclones.

(2) Preparation of Probe

Based on the cDNA nucleotide sequence of rat VIP receptor alreadyreported, primers for PCR were synthesized with a DNA synthesizer (Model391, PCR-MATE EP, Applied Biosystems). ##STR18##

Using 5 μg of rat lung poly(A)⁺ RNA fractions prepared in a mannersimilar to that of the brain RNA fractions and a random primer, cDNAhaving only first strand was synthesized. Then, using this singlestranded DNA as a template, and using the above-mentioned primers, ratVIP receptor cDNA fragments were amplified by the PCR method. Thesequences of the resulting cDNA fragments were determined, and they areconfirmed to be cDNA fragments of rat VIP receptor.

(3) Screening

The λgt11 cDNA library (3×10⁶ pfu) prepared in Example 3 (1) was mixedwith magnesium sulfate-treated E. coli Y1090, and incubated at 37° C.for 15 minutes. Then, 0.5% agarose/LB was added thereto, followed byplating on a 1.5% agar/LB plate. A nitrocellulose filter was placed onthe plate on which a plaque is formed, and the plaque was transferredonto the filter. After alkali treatment of this filter, the DNA wasfixed by heating at 80° C. for 3 hours. This filter was hybridized withthe probe labeled in hybribuffer S 0.2% poly(vinylpyrrolidone), 0.2%bovine serum albumin, 0.2% ficoll 400, 2×SSC and 0.17% yeast RNA)overnight at 55° C. The labeling of the probe was conducted by the useof a multi-prime labeling kit (Amersham). Washing was carried out with2×SSC, 0.1% SDS at 50° C. for 1 hour, and then, hybridized clones weredetected by autoradiography at -80° C. As a result, λRPACAPR18 wasobtained.

Further, the rat brain-derived 5'-extended cDNA library (Clontech)(1.7×10⁶ pfu) was screened, using the cDNA portion of λRPACAPR18 as aprobe, to obtain λRPACAPR46, λRPACAPR5, λRPACAPR12, etc. At this time, abuffer was used which comprised 50% formamide (Bethesda ResearchLaboratories), 5×Denhardt's solution 0.02% bovine serum albumin(Sigma)!, 0.02% poly(vinylpyrrolidone (Sigma), 0.02% ficoll (Sigma),5×SSPE (0.15M sodium chloride, 0.01M monosodium phosphate and 1 mMEDTA), 0.1% SDS and 100 μg/ml of heat-denatured salmon sperm DNA(Sigma), and incubation was conducted overnight at 42° C. together withthe labeled probe to hybridize. Washing was carried out with 2×SSC, 0.1%SDS at 55° C. for 1 hour, and then, hybridized clones were detected byautoradiography at -80° C.

(4) Subcloning of cDNA Clones and DNA Sequence Analysis

An insert portion of the resulting cDNA clone was cut out by cleavagewith EcoRI, and subcloned into plasmid vector pcDNAI or pUC118 to obtainpRPACAPR18 (pcDNAI), pRPACAPR46 (pcDNAI), pRPACAPR5 (pcDNAI) orpRPACAPR12 (pUC118). Further, pRPACAPR46 and pRPACAPR5 were recombinedat the BamHI sites to prepare pRPACAPR46-5. The plasmid was furthercleaved stepwise by exonuclease digestion, or self cyclized or subclonedafter cleavage with an appropriate restriction enzyme (NcoI, PstI orBamHI) to prepare a template DNA for sequence analysis. For sequencedetermination, a fluorescent DNA sequencer (Applied Biosystems) wasused, and for data analysis, a DNASIS (Hitachi Software Engineering) wasused. Results of analysis revealed that pRPACAPR46-5 has the nucleotidesequence of SEQ ID NO: 40, and that pRPACAPR12 has the nucleotidesequence of SEQ ID NO: 41.

Example 4! Screening of Human PACAP Receptor Protein cDNA and DNASequence Analysis

(1) Preparation of Probe

The nucleotide sequence represented by SEQ ID NO: 51 corresponding to acomplementary strand of the N-terminal amino acid sequence having theamino acid sequence represented by SEQ ID NO: 50 of the bovine PACAPreceptor protein obtained in Example 1 was synthesized with a DNAsynthesizer (Model 391, PCR-MATE EP, Applied Biosystems).

(2) Screening

The human pituitary gland-derived cDNA library (λgt11, Clontech)(1.4×10⁶ pfu) was mixed with magnesium sulfate-treated E. coli Y1090,and incubated at 37° C. for 30 minutes. Then, 0.6% agarose/LB was addedthereto, followed by plating on a 1.5% agar/LB+50 μg/ml ampicillinplate. A nitrocellulose filter was placed on the plate on which a plaqueis produced, and the plaque was transferred onto the filter. Afteralkali treatment and neutralization treatment of this filter, the DNAwas fixed by heating at 80° C. for 3 hours. This filter wasprehybridized in a hybridization buffer 7% SDS (Nakarai), 1% bovineserum albumin, 0.5M Na-PO₄ (pH 7.2) and 1 mM EDTA (Dojin)!, and thenhybridized with the probe labeled in the same buffer overnight at 55° C.For the labeling of the probe, terminal labeling was conducted usingγ-³² P!ATP (Du Pont NEN) and T4 kinase (Takara). Washing was carried outtwice with 2×SSC, 0.1% SDS at 55° C. for 30 minutes, and then,hybridized clones were detected by autoradiography at -80° C. As aresult, λ#14 was obtained.

(3) Subcloning of cDNA Clones and DNA Sequence Analysis

An insert portion of the resulting cDNA clone was cut out by cleavagewith EcoRI, and subcloned into plasmid vector pUC118 to obtain pTS847-1.After further cleavage with an appropriate restriction enzyme (SacI,NcoI or HpaI), the plasmid was self cyclized to prepare a template DNAfor sequence analysis. For sequence determination, a Bca Best SequencingKit (Takara) was used, and for data analysis, a DNASIS (Hitachi SoftwareEngineering) was used. Results of analysis revealed that pTS847-1 hasthe nucleotide sequence of SEQ ID NO: 42. Among the nucleotidesequences, the nucleotide sequence coding for mature human PACAPreceptor Type I-A is represented by SEQ ID NO:34. The deduced amino acidsequence of human PACAP receptor Type I-A is represented by SEQ IDNO:23.

(4) Preparation of a primer for PCR based on the nucleotide sequence ofhuman PACAP receptor Type I-A

A region into which the insertion region of human PACAP receptor beingdeduced to enter was amplified by PCR. Primers of following SEQ ID NO:52and SEQ ID NO:53 were prepared based on the nucleotide sequence ofpTS847 coding for human PACAP receptor Type I-A obtained in Example4(3). Sequence:HPRF 5'CTGGGATATGAATGACAGCACAGC 3' (SEQ ID NO:52; anucleotide sequence of 1132nd to 1155th of pTS847) Sequence:HPRR5'TCTGGGGAGAAGGCAAATACTGTG 3' (SEQ ID NO:53; a complementary nucleotidesequence of 1342nd to 1355th of pTS847)

(5) Application of PCR on human pituitary and amigdaloid nucleus

Two(2) ng of cDNA of human pituitary and amigdaloid nucleus (Quick-ClonecDNA, Clonetech) and each 0.5 μM of primers obtained Example 4(4), each10 mM of dNTP were mixed in a PCR reaction buffer, and Taq polymerasewas added thereto. Denaturing was conducted at 94° C. for 45 seconds,anealing was held at 60° C. for 45 seconds and elongation reaction washeld at 72° C. for 2.5 minutes to obtain PCR product.

(6) Subcloning of PCR product and DNA sequence analysis

The resulting PCR product was inserted into Hinc II site of a plasmidpUC118 and was subjected to a subcloning. Of the clones subcloned,Southern blotting was conducted to screen subtypes. In order to screen aclone of human PACAP receptor Type I-B, the following probe of SEQ IDNO:54 was synthesized based on the sequence of the insertion region ofrat PACAP receptor Type I-B. 5'TGCGTGCAGAAATGCTACTGCAAGCCACAG 3' (SEQ IDNO:54)

In order to screen a clone of human PACAP receptor Type I-C, thefollowing probe of SEQ ID NO:54 was synthesized based on the sequence ofthe insertion region which is different from Type I-B which was reportedin rat (Nature, 365, p170-175, 1993). 5'GACCCCCTGCCTGTGCCCTCAGACCAGCAT3' (SEQ ID NO:55)

Clones of pHPR15A and pHPR55A were obtained from the Southern blot ofSEQ ID NO:54 and a clone of pHRP66P was obtained from the Southern blotof SEQ ID NO:55 (FIG. 15). Dideoxy method using RI labelled dCTP wasemployed for the determination of the nucleotide sequences of theseclones. DNASIS (Hitachi Soft Engineering Co. Ltd.) was used for analysisof the data. The nucleotide sequences of cDNA coding for human PACAPreceptor Type I-B, Type I-B2 and Type I-C and the amino acid sequencesdeduced therefrom are shown in FIGS. 16, 17 and 18, respectively. Thenucleotide sequences of cDNA coding for human PACAP receptor Type I-B,Type I-B2 and Type I-C are represented by SEQ ID NO:35, SEQ ID NO:36 andSEQ ID NO:37, respectively. The amino acid sequences deduced therefromare represented by SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29,respectively.

Example 5! Expression of Bovine PACAP Receptor Protein cDNAs

(1) Preparation of Transformants Containing Bovine PACAP ReceptorProtein cDNAs

cDNA clone pBPR35 of the bovine PACAP receptor protein obtained inExample 2 was digested with SmaI and BamHI, thereby cutting out afragment (about 800 bp) from the plasmid. Then, a HindIII linker wasadded to the SmaI-digested terminus of this fragment. The resultingfragment was named "fragment A". On the other hand, two kinds offragments were obtained by digesting pBPR68 with BamHI and SmaI. One ofthem, a fragment of about 1 kbp (named "fragment B"), was cut out. Thesefragment A and fragment B were ligated with each other at the respectiveBamHI-digested sites to prepare recombinant cDNA (pBPR-T). pBPR-T wasinserted in the HindIII and EcoRV sites downstream of a CMV promoter ofexpression vector pRc/CMV to prepare an expression vector. Thisexpression vector was introduced into a CHO cell by the calciumphosphate method using a CellPhect transfection kit (Pharmacia) toobtain a transformant. The transformant cells were selected with 500μg/ml G-418 (trade mark: Geneticin, Lifetech Oriental).

(2) Preparation of Membrane Fraction of the Transformants

The transformants (CHO cells) cultivated for 3 days after subculturewere separated using 0.2 mM EDTA/phosphate buffer, and suspended in 10mM sodium carbonate buffer supplemented with 1 mM EDTA, 0.5 mMphenylmethylsulfonyl fluoride (PMSA), 20 μg/ml leupeptin, 4 μg/ml E-64and 1 μg/ml pepstatin. The suspended cells were disrupted with aPolytron homogenizer (Kinematica). The disrupted product was centrifugedwith a high speed cooling centrifuge (CR26H, Roter RR18, Hitachi, Ltd.)at 3,000 rpm for 10 minutes to obtain a supernatant. The resultingsupernatant was further ultracentrifuged with an ultracentrifuge(SCP70H, Roter RPZ35T, Hitachi, Ltd.) at 30,000 rpm for 60 minutes toobtain pellets. The resulting pellets were suspended in a buffer 20 mMTris-HCl (pH 7.4), 0.25M sucrose, 2 mM EDTA, 0.5 mM PMSF, 20 μg/mlleupeptin and 1 μg/ml pepstatin! to prepare a membrane fractionsuspension.

(3) Saturation Binding Experiment of Bovine PACAP Receptor ProteincDNA-Expressed CHO Cells

The membrane fraction of the transformants obtained in (2) describedabove was reacted with 100 pM ¹²⁵ I!-PACAP27 in a buffer 20 mM Tris-HCl(pH 7.4), 5 mM magnesium acetate, 2 mM EGTA, 0.5 mM PMSF, 20 μg/mlleupeptin, 4 μg/ml E-64 and 1 μg/ml pepstatin! at 25° C. for 75 minutes.Bound ligands were separated from free ligands through a glass fiberfilter. The non-specific binding was examined in the presence of 1 μMPACAP27 (FIG. 33). The binding was examined with a a γ-ray counter. Thedissociation constant and the maximum binding were examined by Scatchardplot analysis (FIG. 34).

(4) Competitive Binding Experiment of Bovine PACAP Receptor ProteincDNA-Expressed CHO Cells

Under the conditions of the binding experiment of (3) described above,PACAP27, PACAP38 and VIP were added to examine competition with ¹²⁵I!-PACAP27. Bovine PACAP receptor protein on the membrane fractionshowed a high reactivity, but low in reactivity with VIP (FIG. 35).

(5) Assay of Intracellular Cyclic AMP Production of Bovine PACAPReceptor Protein cDNA-Expressed CHO Cells

Three days after the transformants (CHO cells) were plated on a 24-wellplate, the cells were washed with Hank's buffer (composition: 8 g/lNaCl, 0.4 g/l KCl, 0.06 g/l Na₂ HPO₄, 1.0 g/l glucose, 0.2 g/l MgSO₄,0.14 g/l CaCl₂ and 0.35 g/l NaHCO₃) supplemented with 0.05% BSA, andtreated in the presence of 0.2 mM 3-isobutyl-1-methylxanthine at 37° C.for 1 hour. PACAP27, PACAP38 and VIP of various concentrations wereadded thereto, followed by cultivation at 37° C. for 30 minutes. Afterthe cells were washed with the above-mentioned Hank's buffersupplemented with 0.05% BSA, intracellular cyclic AMP was extracted bythe use of 500 μl of Hank's buffer and 100 μl of 20% perchloric acid,and neutralized with 1.5M KOH. The amount of cyclic AMP was assayed witha cAMP oxygen immunoassay system (BIOTRAK Amersham). The concentrationof intracellular cyclic AMP increased depending on the concentrations ofPACAP27 and PACAP38 (FIG. 36).

(6) Assay of Intracellular Inositol Phosphate of Bovine PACAP ReceptorProtein cDNA-Expressed CHO Cells

The pathway of signal transmission of inositol phosphate well known asthe pathway of signal transmission together with cyclic AMP wasexamined. Three days after the transformants (CHO cells) were plated ona 24-well plate, 5 μCi myo- ³ H! inositol (19.1 Ci/mmole, Amersham) wasadded to the cell culture solution, followed by cultivation overnight at37° C. The cells were washed with an assay buffer (20 mM HEPES, 140 mMNaCl, 4 mM KCl, 1 mM Na₄ HPO₄, 1 mM MgCl₂, 1.25 mM CaCl₂, 10 mM LiCl, 10mM glucose and 0.1% BSA). Then, PACAP27, PACAP38 and VIP of variousconcentrations were added to 500 μl of the assay buffer, and themixtures were added to plates, followed by reaction with the cells at37° C. for 20 minutes. One hundred microliters of 20% perchloric acidwas added thereto to stop the reaction, and intracellular inositolphosphate was extracted. The extract was neutralized with 1.5M KOH. Allinositol phosphate was separated from free inositol by the use of ionexchange chromatography (AGI-X8, Bio RAD). Thereafter, inositolphosphate was eluted with 1M ammonium formate/0.1M formic acid, and theamount of inositol phosphate was measured with a scintillation counter.The concentration of inositol phosphate increased depending on theconcentrations of PACAP27 and PACAP38 (FIG. 37).

Example 6! Confirmation of Expression Site of Rat PACAP Receptor mRNA

(1) Preparation of Poly(A)⁺ RNA

Total RNAs were prepared from the brains, lungs, livers, kidneys andtestes of 8-week-old Sprague Dawley rats (males, Nippon Charles River)by the guanidine isothiocyanate method Biochemistry, 18, 5294 (1979) andMethod in Enzymology, 154 3 (1987)! and poly(A)⁺ RNA prepared from thebrains, lungs, livers, kidneys and testes was fractionated byformalin-modified agarose gel electrophoresis Proc. Natl. Acad. Sci.U.S.A., 77, 5794 (1980)! contained 2.2M formalin (Wako Pure ChemicalIndustries), followed by transfer to a nylon membrane filter (Pole).

(2) Preparation of Probe

374-bp fragment haveing the nucleotide sequence from the 76th to 450thof DNA (rat PACAP receptor cDNA pRPACAPR12) represented by thenucleotide sequence of SEQ ID NO:41 was labeled with ³² P by the use ofa multi-prime labeling kit (Amersham) to prepare a probe.

(3) Northern Hybridization

The filter of (1) described above was treated at 80° C. for 2 hours tofix RNA, followed by hybridization in a hybridization buffer 50%formamide deionized, 5×SSPE, 5×Denhardt's solution, 0.5% SDS, and 100μg/ml heterologous salmon sperm DNA heat denatured after ultrasonication(Wako Pure Chemical Industries)! overnight at 42° C. Subsequently, theprobe obtained in (2) described above was heat denatured, and theheat-denatured probe was added thereto, followed by hybridizationovernight at 42° C. Washing was conducted 5 times with 2×SSC, 0.1% SDSat 55° C. for 30 minutes, and further twice 0.1×SSC, 0.1% SDS at 50° C.for 20 minutes. Autoradiography was carried out for 12 hours using animage analyzer (Fuji BAS-2000) to detect desired bands. Results thereofrevealed that PACAP receptor mRNA was most expressed in the brains, thatexpression thereof was also observed in the lungs and the livers, andthat the size of mRNA was about 6.5 kb (FIG. 38).

Example 7! Expression of Rat PACAP Receptor Protein cDNA

(1) Construction of Expression Vector for Animal Cells of Rat PACAPReceptor Protein cDNA

Using plasmids pRPACAPR46-5 and pRPACAPR12 obtained in Example 3, anNcoI fragment having an N-terminal translation initiation codon wasprepared. After repair of both ends of this fragment with Klenowfragments (Takara), HindIII linkers (Takara) were added thereto, andfurther cleaved with BamHI. Of the resulting fragments, a fragmentcontaining the translation initiation codon was recovered byelectrophoresis, and ligated with cDNA I obtained by cleaving BamHI-ApoIfragments of pRPACAPR46-5 and pRPACAPR12 with HindIII and EcoRI,respectively, to construct an expression vector in which NcoI-ApoIportions of the respective cDNA fragments were inserted. These plasmidswere further cleaved double by the use of HindIII and XbaI, and DNAfragments containing cDNA portions were incorporated into other animalcell expression vectors, pRc/CMV, utilizing the same sites, to obtainexpression vectors pRPR3-A (derived from pRPACAPR46-5) and pRPR4-B(derived from pRPACAPR12).

(2) Introduction of Expression Vector into CHO Cells 9.0×10⁵ CHO cellswere subcultured to each tissue culture flask having a bottom area of 25cm² (Corning), and cultivated for 24 hours in a culture solution(culture solution A) composed of Ham's F12 medium (Flow), 10% fetalbovine serum, and penicillin and streptomycin as antibiotics. Expressionvectors pRPR3-A (derived from pRPACAPR46-5) and pRPR4-B (derived frompRPACAPR12) obtained in (1) described above were introduced into CHOcells each in an amount of 10 μg with a gene introduction kit(CellPhect, Pharmacia) by the calcium phosphate method according to theformulation of the kit. After 24 hours, the culture solution wasexchanged. After further 24 hours, the solution was-exchanged by culturesolution A supplemented with 500 μg/ml of G418, and cDNA-introducedcells were selected, based on resistance to G418.

(3) Binding Experiment of PACAP Receptor Protein and ¹²⁵ I!-PACAP27 onCHO Cell Membrane

CHO cells exhibiting resistance to G418 were recovered by trypsindigestion, and subcultured to a 12-well plate for tissue culture. Thecells were incubated until they covered the bottom surface of the tissueculture plates completely. Untreated CHO cells and rat VIP receptorcDNA-introduced CHO cells were also similarly cultivated. The cells werewashed twice with a buffer for the binding experiment Hank's solution(pH 7.4) containing 5 mM HEPES, 5% CHAPS and 0.1% BSA!. Then, the bufferand ¹²⁵ I!-PACAP27 were successively added so as to give a final ¹²⁵I-PACAP27 concentration of 100 pM. The amount of the reaction solutionper well was 500 μl. and the radioactivity was about 11.4×10⁴ cpm. Foranalysis of specificity, samples containing unlabeled PACAP27 of a finalconcentration of 1 μm and VIP, in addition to the samples containingonly the labeled products, were prepared. After incubation at 37° C. for1 hour, the cells washed three times with the buffer for the bindingexperiment were dissolved with 1 ml of 0.5N NaOH and 0.1% SDS for eachwell, and the radioactivity contained therein was measured with aγ-counter. Results of measurements are shown in FIG. 39. Columns 1 to 12in FIG. 39 indicate the radioactivity in CHO cells under the followingconditions:

Column 1: untreated CHO cells+ ¹²⁵ I!-PACAP27

Column 2: untreated CHO cells+ ¹²⁵ I!-PACAP27+cold PACAP27

Column 3: untreated CHO cells+ ¹²⁵ I!-PACAP27 +cold VIP

Column 4: pRPR3-A-introduced CHO cells+ ¹²⁵ I!-PACAP27

Column 5: pRPR3-A-introduced CHO cells+ ¹²⁵ I!-PACAP27+cold PACAP27

Column 6: pRPR3-A-introduced CHO cells+ ¹²⁵ I!-PACAP27+cold VIP

Column 7: pRPR4-B-introduced CHO cells+ ¹²⁵ I!-PACAP27

Column 8: pRPR4-B-introduced CHO cells+ ¹²⁵ I!-PACAP27+cold PACAP27

Column 9: pRPR4-B-introduced CHO cells+ ¹²⁵ I!-PACAP27+cold VIP

Column 10: rat VIP receptor cDNA-introduced CHO cells+ ¹²⁵ I!-PACAP27

Column 11: rat VIP receptor cDNA-introduced CHO cells+ ¹²⁵ I!-PACAP27+cold PACAP27

Column 12: rat VIP receptor cDNA-introduced CHO cells+ ¹²⁵ I!-PACAP27+cold VIP

FIG. 39 indicates that the radioactivities in the pRPR3-A-introduced CHOcells and the pRPR4-B-introduced CHO cells (column 4 and column 7,respectively) are higher than that in the untreated CHO cells (column1). This fact proved that each of the pRPR3-A-introduced CHO cells andthe pRPR4-B-introduced CHO cells produced PACAP receptors.

(4) Analysis of Specificity of Rat PACAP Receptor on CHO Cell MembraneUsing ¹²⁵ I!-PACAP27

The pRPR3-A-introduced and pRPR4-B-introduced CHO cells obtained in (2)described above were each disrupted in sodium carbonate buffercontaining 1 mM EDTA, 0.5 mM PMSF, 20 μg/ml leupeptin, 4 μg/ml E-64 and1 μg/ml pepstatin with a Polytron homogenizer (Kinematica) to preparemembrane fractions. Using the membrane fractions, complex bindingexperiments were conducted. For each of the membrane fractions of thepRPR3-A-introduced CHO cells and the pRPR4-B-introduced CHO cells, eachof 10 μg and 15 μg (converted to a protein amount) thereof was ligatedwith 100 pM of ¹²⁵ I!-PACAP27 in a buffer containing 20 mM Tris (pH7.4), 1 mM EDTA, 0.05% CHAPS, 0.1% BSA and various protease inhibitors.For the competitive experiments, PACAP27 and VIP having eachconcentration were added. The reaction was conducted at 25° C. for 1hour, and bound ligands were separated from free ligands by filtrationthrough a filter. As to non-specific binding, a value in the case that 1μM unlabeled PACAP27 was added and used as a standard. The amount ofbound ligands was measured with a γ-counter. After elimination of thenon-specific binding, it was examined whether or notconcentration-dependent competition took place. Results thereof revealedthat concentration-dependent competition took place. For VIP similar toPACAP27 in structure, competition was observed only at a concentrationmuch higher than that of PACAP27, which showed that the PACAP receptorprotein which was allowed to express was PACAP-specific (FIG. 40).

(5) Screening of Clones Highly Producing Rat PACAP Receptor Protein byBinding Experiment with ¹²⁵ I!-PACAP27

The rat PACAP receptor protein cDNA-introduced CHO cells obtained in (2)described above were each subcultured to 10-cm diameter dishes at a lowdensity. After cultivated until formation of colonies, each of thecolonies was dispersed and recovered by suction. Cells derived from therespective colonies were separately subcultured in 6-well plates fortissue culture, followed by binding experiments using parts thereof in amanner similar to that of (4) described above (FIG. 41). Clones havingrelatively more bound ¹²⁵ I!-PACAP27 when compared among wells wereselected, and the reproducibility was further confirmed. As a result,clones A12 and B17 reproducibly binding to ¹²⁵ I!-PACAP27 much more wereselected from the pRPR3-A-introduced pRPR4-B-introduced CHO cells (FIG.42).

(6) Assay of Intracellular Cyclic AMP of Rat PACAP Receptor ProteincDNA-Introduced CHO Cells

From the binding experiment with ¹²⁵ I!-PACAP27 of (3) described above,using CHO strains A12 and B17 highly producing rat PACAP receptorprotein, the production promotion of intracellular cyclic AMP withPACAPs was detected in the following manner. Each 48-well plate fortissue culture was inoculated with each of the cell strains at a densityof 1.0×10⁵ cells/well, followed by cultivation for 3 days. The plate waswashed twice with Ham's F12 medium supplemented with 0.1% BSA and 0.5 mMIBMX, and 500 μl/well of the same medium was added thereto. PACAP27,PACAP38 or VIP having each concentration was added thereto in a 1/100amount, followed by standing at 37° C. for 40 minutes. The supernatantwas removed, and extraction was conducted with 100% cold ethanol. Theextract was evaporated to dryness with a centrifugal freeze dryer, andredissolved in the buffer attached to an EIA kit for assaying cyclic AMP(Amersham). Then, the amount of cyclic AMP was assayed according to theformulation of the kit. Results thereof revealed that both A12 and B17promoted the production of intracellular cyclic AMP, depending on theconcentrations, for PACAP27 and PACAP38, but a concentration much higherthan that of the PACAPs was required to promote the production ofintracellular cyclic AMP, for VIP (FIG. 43).

(7) Construction of Rat PACAP Receptor Protein cDNA Expression SystemUsing Baculovirus

Animal cell expression vectors pRPR3-A and pRPR4-B were each cleavedwith HindIII, and the termini were repaired with Klenow fragments(Takara), followed by addition of BglII linkers. The resulting fragmentswere further cleaved with XbaI, and the termini were repaired withKlenow fragments, followed by addition of HindIII linkers. These DNAswere each digested double by the use of HindIII and BalII, therebyobtaining DNA fragments corresponding to translation regions.pBlneBacIII, a baculovirus transfer vector, was similarly digesteddouble with HindIII and BglII, and subjected to ligation reaction withthe above-mentioned DNA fragments. According to the formulation of a kit(Maxbac baculovirus expression system, Invitrogen) with which plasmidDNA confirmed in insertion was prepared, the resulting fragments,together with baculovirus genome DNA, were introduced into Sf9 cells.After cultivation at 27° C. for 2 days, virus particles appeared in thesupernatant were recovered. Recombinant viruses were selected therefromby the plaque assay in accordance with the formulation of the kit.

(8) Expression Using Rat PACAP Receptor Protein cDNA-IntroducedBaculovirus

The recombinant plaques formed by the plaque assay were extracted with amicropipette, and dispersed in 1 ml of complete medium for Sf9 (Gracemedium for insects (Gibco) containing necessary additives, inactivatedcalf serum and gentamicin!. A 25-cm² flask for tissue culture wasinoculated with 2×10⁶ Sf9 cells, together with 5 ml of the medium, andthe cells were adhered to a bottom of the flask, followed by addition of500 μl of the above-mentioned virus solution. After cultivation at 27°C. for 5 days, the cells were recovered by pipetting. The cells werepelletized by centrifugation, and suspended in a small amount of medium.Then, a 1/10 amount of the suspension was poured into each Eppendorftube. After further centrifugation, the supernatant was replaced by thesame buffer as with the binding experiment in animal cells (composition:Hank's solution (pH 7.4) containing 5 mM HEPES, 5% CHAPS and 0.1% BSA!.Then, ¹²⁵ I!-PACAP27 was added so as to give a final concentration of100 pM, and unlabeled PACAP27 of a final concentration of 1 μM was addedto a sample for analysis of specificity to make up a total solutionamount of 500 μl. After standing at room temperature for 1 hour andbinding, bound ligands, together with the cells, were pelletized bycentrifugation. The pellets were further resuspended in the same bufferand centrifuged. After this procedure was repeated three times toconduct sufficient washing, the amount of radioligands remaining in thepellets was measured with a γ-counter. As a result, 4 virus clonesshowing a very high binding were obtained (FIG. 44).

Example 8! Expression of Human PACAP Receptor Protein cDNA

(1) Preparation of Transformant in Baculovirus System Using Human PACAPReceptor Protein cDNA

A fragment cut out by digestion with BamHI and PstI from animal cellexpression vector pcDNAI/Amp in which human PACAP receptor protein wassubcloned was inserted in the BamHI and PstI sites of transfer vectorpBlueBacIII to prepare a recombinant transfer vector. Sf9 cells weretransfected with this vector, together with baculovirus DNA (AcMAPVDNA), using the transfection module attached to the kit (MAXBAC,Inbitrogen). After transfection, the viruses appeared in thesupernatant, so that the culture supernatant of the fourth day was usedas a virus solution. Sf9 cells (2×10⁶ cells) seeded on a 6-cm² dish wereinfected with this virus solution at room temperature for 30 minutes,and a medium containing 0.6% agarose was poured therein for fixing. Cellculture at a high humidity (a humidity of 100%) for 5 to 6 days resultedin development of virus plaques. Plaques caused by viruses in whichhuman PACAP receptor protein was recombined could be judged by turningblue, and the viruses were recovered. The recombinants were purified byrepetition of this plaque assay. Sf9 cells were infected with thepurified recombinants, and cultivated for 48 to 72 hours, whereby PACAPreceptor protein-expressed transformants could be obtained (FIG. 45).

(2) Construction of Cell Strain Expressing Human PACAP Receptor

An overall length fragment was cut out from human PACAP receptorcDNA-cloned pTS847-1 by digestion with EcoRI, and inserted in the EcoRIsite of animal cell expression vector pRc/CMV so as to be arranged in acorrect direction, thereby constructing pTS849. The resulting plasmidswere introduced into CHO-K1 cells (ICN) by the calcium phosphate method,and plasmid-incorporated clones were selected with 500 μg/ml G-418(Geneticin).

(3) Scatchard Plot Analysis Using Membrane Fraction of Human PACAPReceptor Protein-Expressed CHO-K1 Cells and Competitive InhibitionAnalysis

The human PACAP receptor-expressed CHO-K1 cells obtained in (2)described above were cultivated in ten 175-cm² flasks containing amedium supplemented with 500 μg/ml G-418 (trade mark: Geneticin,Lifetech Oriental). When the cells covered almost entire bottom surfacesof the flasks, the CHO-K1 cells were separated with PBS solutioncontaining 1 mM EDTA. After washing with the same buffer, the CHO-K1cells were suspended in 10 mM NaCO₃ buffer containing 1 mM EDTA, 0.5 mMPMSF, 20 μg/ml leupeptin, 20 μg/ml E-64 and 1 μg/ml pepstatin, anddisrupted with a Polytron homogenizer (Kinematica). Then, the disruptedproduct was centrifuged with a high speed cooling centrifuge (CR26H,Roter RR18, Hitachi, Ltd.) at 3,000 rpm for 10 minutes. The resultingsupernatant was further ultracentrifuged with an ultracentrifuge(SCP70H, Roter RP42, Hitachi, Ltd.) at 30,000 rpm for 60 minutes. Theresulting pellets were suspended in a buffer containing 20 mM Tris-HCl(pH 7.4), 0.25M sucrose, 2 mM EDTA, 0.5 mM PMSF, 20 μg/ml leupeptin and1 μg/ml pepstatin. The resulting suspension was used as a membranefraction.

The preparation of ¹²⁵ I!-PACAP27, the Scatchard plot analysis obtainedfrom the saturation binding experiment, and the competitive inhibitionexperiment were carried out in accordance with the method described inExample 1 (3).

From results of Scatchard plot analysis, a single binding site existedin the membrane fraction of the human PACAP receptor protein-expressedCHO-K1 cells, and the dissociation constant (Kd) was 41±6.9 pM (FIG.46). Further, results of the competitive inhibition experiment provedthat PACAP27 and PACAP38 competed with ¹²⁵ I!-PACAP27. On the otherhand, it was revealed that VIP was 1,000 times weaker than PACAP27 (FIG.47).

(4) Assay of Intracellular Cyclic AMP of Human PACAP ReceptorProtein-Expressed CHO-K1 Cells

The human PACAP receptor protein-expressed CHO-K1 cells obtained in (2)described above were cultivated in a 24-well plate containing a mediumsupplemented with 500 μg/ml G-418 (trade mark: Geneticin, LifetechOriental) until the cells almost covered an entire surface of the plate.After washing twice with Hank's buffer containing the culture buffer, 10mM HEPES and 0.05% BSA, the CHO-K1 cells were cultivated in theabove-mentioned buffer supplemented with 0.2 mM3-isobutyl-1-methylxanthine at 37° C. for 60 minutes. Then, PACAP27,PACAP38 or VIP having each concentration was added thereto, followed byfurther cultivation at 37° C. for 30 minutes. After absorption of thebuffer, the cells were washed twice with the culture buffer. Then, cAMPwas extracted from the cells with 20% perchloric acid. After transfer toa 1.5-ml Eppendorf tube, the extract was centrifuged with a Tomymicrocentrifuge at 12,000 rpm for 5 minutes, and the supernatant wasneutralized with 1.5N KOH/60 mM HEPES to prepare a cell eluted solution.The concentration of cyclic AMP was determined by the acetylation methodof a cAMP assay system (Amersham). Under these determination conditions,when nothing was added, the amount of intracellular cAMP was 0.7pmole/well. For PACAP27 and PACAP38, the concentration of intracellularcAMP increased depending on the concentrations. In particular, when 0.1mM of PACAP38 was added, accumulation of cyclic AMP about 30 times thebasal level (about 21 pmoles/well) was observed (FIG. 48). VIP littleraised the concentration of intracellular cyclic AMP, compared withPACAP27 and PACAP38 (FIG. 48).

Example 9! Expression of Human PACAP Receptor mRNA

Poly(A)⁺ RNA (Clontech) from each human tissue was subjected to 1.1%agarose gel-modified gel electrophoresis containing 2.2M formalin forfractionation, followed by transfer to a nylon membrane filter. Then,RNA transferred was fixed to the nylon membrane with UV. A probe ofhuman PACAP receptor cDNA (SacI-BglII fragment of pTS847-1, nucleotideNo. 168-562) was prepared with a random prime labeling kit (Amersham)and α-³² P!dCTP (Du Pont/NEN), and northern hybridization was carriedout using this probe. As a result, human PACAP mRNA was most expressedin the brain, and the size thereof was about 7 kb. Expression was alsoobserved in the lung, the liver, the pancreas and other organs, althoughweak (FIG. 49).

Example 10! Expression of PACAP mRNA in Rat Central Nerve System

All RNAs were prepared from the olfactory bulbs, amygdalae, cerebralbasal ganglia, hippocampi, thalami, hypothalami, cerebral cortices,medulla oblongatas, cerebellums, spinal cords and pituitary glands of8-week-old S. D. rats (♂) by the guanidine isothiocyanate method, andpoly(A)⁺ RNA was further prepared by the use of an oligo(dT) spun-column(Pharmacia). Five micrograms of poly(A)⁺ RNA prepared from the aboveregions of the central nervous system was fractionated by 1.2%formalin-modified agarose gel electrophoresis Proc. Natl. Acad. Sci.U.S.A., 77, 5794 (1980)! contained 2.2M formalin (Wako Pure ChemicalIndustries), followed by transfer to a nylon membrane filter (Pole).

(2) Preparation of Probe

374-bp fragment having the nucleotide sequence from the 76th to 450th ofDNA (rat PACAP receptor cDNA pRPACAPR12) represented by the nucleotidesequence of SEQ ID NO: 41 was labeled with ³² P by the use of amulti-prime labeling kit (Amersham) to prepare a probe.

(3) Northern Hybridization

The filter of (1) described above was treated at 80° C. for 2 hours tofix RNA, followed by hybridization in a hybridization buffer 50%formamide deionized, 5×SSPE, 5×Denhardt's solution, 0.5% SDS, and 100μg/ml heterologous salmon sperm DNA heat denatured after ultrasonication(Wako Pure Chemical Industries)! overnight at 42° C. Subsequently, theprobe obtained in (2) described above was heat denatured, and theheat-denatured probe was added thereto, followed by hybridizationovernight at 42° C. Washing was conducted 5 times with 2×SSC, 0.1% SDSat room temperature for 5 minutes, and further twice 0.1×SSC, 0.1% SDSat 50° C. for 20 minutes. Autoradiography was carried out for 7 daysusing a X-OMAT AR film (Kodak) to detect desired bands.

Results thereof revealed that rat PACAP receptor mRNA was expressed inalmost all regions of the central nerve system, and that there waslittle expression in the cerebellums and pituitary glands (FIG. 50).From these results, the PACAPs are deduced to play an important role inthe central nerve system.

Example 11! Screening of Human PACAP Receptor Antagonist Which Uses CellMembrane Fraction of Sf9 cell Expressing cDNA of Human PACAP ReceptorProtein

(1) Preparation of Buffer for Assay

Composition of buffer

20 mM Tris-HCl, 2 mM EGTA, 5mM (CH₃ COO)₂ Mg.4H₂ O, 0.5 mM PMSF, 1 μg/mlpepstatin, 20 μg/ml leupeptin, 4 μg/ml E-64, 0.03% NaN₃, 0.1% BSA, 0.05%CHAPS, pH7.2

Method for Preparation

The agents other than peptidase inhibitor (PMSF, pepstatin, leupeptin,E-64) and BSA were dissolved into distilled water. While controlling pHof the aqueous solution with 6N HCl, peptidase inhibitor was addedthereto. Pepstatin and PMSF were dissolved into DMSO and the DMSOsolution was added to the distilled water solution with rapid agitation.Final concentration of DMSO was adjusted to 0.1%, thus pepstatin andPMSF were dissolved into 1 ml of DMSO to prepare 1 liter of buffer. Thenthe solution was mixed and BSA was added thereto.

(2) Sf9 cells which express human PACAP receptor protein obtained inExample 8 were disrupted by Polytron mixer in a buffer for homogenize(20 mM Tris-HCl, 2 mM EDTA, 0.5 mM PMSF, 1 μg/ml pepstatin, 20 μg/mlleupeptin, 4 μg/ml E-64, pH7.4). The disrupted cell solution wascentrifuged at 3,000 rpm for 5 minutes and the supernatant wascentrifuged at 30,000 rpm for 60 minutes. The resulting precipitate wastreated as a membrane fraction. The membrane fraction was diluted withthe buffer for assay to 2 μg protein/ml. The diluted solution wasapplied on a cell strainer (FALCON, 2350) and was divided into 100 μl ineach tube (FALCON, 2053) with dispenser.

(3) Each 1 μl of 10 mM of the sample was added to the reaction tubes(final concentration: 100 μM, room temperature). DMF was added theretofor assay of the maximum binding amount, and 1 μl of DMF with 10 μMPACAP27 was added for assay of nonspecific binding amount (finalconcentration: 1 μM). The maximum binding amount was assayed twicerespectively at the beginning and the end of the assay, and thenonspecific binding amount was assayed twice at the end of the assay.

(4) In radio isotope region, each 2 μl of 5 nM ¹²⁵ I!-PACAP27 (DuPont)was added in the reaction tubes (final concentration: 100 pM). ¹²⁵I!-PACAP27 was placed on ice.

(5) The reaction tubes were incubated at 25° C. for 1 hour.

(6) 1.5 ml of a detergent buffer was added into the reaction tubes andthe mixture was filtered on a glass fiber paper (Whatman, GF/F) usingSampling manifold (Millipore). 1.5 ml of a detergent buffer was furtheradded to the reaction tubes and they were filtered. The glass fiberfiltration paper (Whatman, GF/F) was previously immersed in a PEI(polyethyleneimine) solution (20 mM Tris-HCl, 0.3% PE1, pH7.4). Thedetergent buffer may be similar with the assay buffer but it is notnecessary to contain. peptidase inhibitor.

(7) ¹²⁵ I! remaining on the glass fiber filtration paper was counted byγ-counter. Based on the counts, inhibiting activity on a bindingspecificity Percent Maximum Binding! of PACAP27 and a PACAP27 receptorof the samples were determined according to the following formula:

    PMB= (B-NSB)/B.sub.0 -NSB)×100

PMB: percent Maximum Binding

B: value when the samples are added,

NSB: non-specific binding amount

B₀ : Maximum Binding

As a result, substances No. 1 to 10 as shown in FIG. 51 were obtained assubstances which inhibited a specific binding of PACAP27 and PACAPreceptor. PMB of the compounds are shown in Table 3.

                  TABLE 3    ______________________________________    Test compound No.                   Specific Binding %    ______________________________________    1              57    2              11    3              37    4              15    5              2    6              -3    7              50    8              15    9              20    10             34    ______________________________________

Example 12! Preparation of Anti-PACAP Receptor Antibody

(1) Preparation of a Partial Peptide of PACAP Receptor

The 5th Cys(C) of the amino acid sequence, MESDCIFKKEQAMC, wassubstituted with Ala(A) for the convenience of a preparation ofimmunoantigen complexes to obtain a partial peptide, MHSDAIFKKQAMC, witha conventional method using a autosynthesizer (430A, AppliedBiosystem).The first amino acid sequence corresponds to 1st to 14th amino acidsequence of SEQ ID NO:14, which is a common sequence to bovine, rat orhuman PACAP receptor represented by the amino acid sequence of anyone ofSEQ ID NO:14 to SEQ ID NO:29.

(2) Preparation of Immunogen

A complex of the synthetic peptide (MHSDAIFKKEQAMC) obtained in theabove (1) and bovine thyroglobulin (BTG) was made and used as animmunogen. Thus, 21 mg of BTG was dissolved into 1.4 ml of 100 mMphosphate buffer (pH 6.8) and the solution was mixed with 2.35 mg ofGMBS in 100 μl of DMF to react at room temperature for 40 minutes. Thereactant was applied on Sephadex G-25 column™ (1×35 cm) equibilliatedwith 100 mM phosphate buffer and to obtain a fraction containing BTG. Ahalf (1.5 ml) of the fraction was mixed with 2 mg of the synthesizedpeptide dissolved in 50% DMSO to react at 4° C. for two days. Thereactant was dialyzed against physiological saline at 4° C. for two daysand the dialyzate was divided into small amount and freeze-restored.

(3) Immunization

100 μg of the immunogen obtained in the above (2) was subcutanouslygiven with a complete Freund's adjuvant to each female BALB/c mouse of 6to 8 week old. Once or twice additional immunizations was conducted atthree weeks intervals.

(4) Preparation of HRP-labelled partial peptide of receptor

HRP (Horse radish peroxidase)-labelled partial peptide necessary forassay for antibody value with EIA was prepared as follows:

Twenty (20) mg of HRP was dissolved into 1.5 ml of phosphate buffer(pH6.5) and the solution was mixed with 1.4 mg of GMBSN-(6-maleimidebutylyloxy)succinimide! in 100 μl of DMF to react at roomtemperature for 40 minutes. The reactant was applied on Sephadex G-25column™ (1×35 cm) equibilliated with 100 mM phosphate buffer and toobtain a fraction containing BTG. A half (1.5 ml) of the fraction wasmixed with 2 mg of the synthesized peptide dissolved in 50% DMSO toreact at 4° C. for two days. The reactant was applied on Ultrogel AcA44column™ (1×35 cm) equibilliated with 100 mM phosphate buffer and toobtain a fraction containing HRP-labelled partial peptide. BSA(finalconcentration: 0.1%) and thimerosal (final concentration: 0.05%) wereadded to the fraction to be restored at 4° C.

(5) Assay of Antibody Titer

Antibody titer of antiserum of the mice immunized in the above (3) wasassayed as follows:

100 μl of 100 μg/ml anti-mouse immuno globulin antibody (IgG fraction,Cuppel) dissolved in 100 mM carbonate buffer (pH 9.6) was added to a96-well plate and kept at 4° C. for 24 hours to make an anti-mouseimmunoglobulin bound microplate. After the plate was washed withphosphate bufferized physiological saline (PBS, pH 7.4), 300 μl ofBlockace (Yukizirushi, Japan) diluted to 25% with PBS was added to theplate to react at 4° C. for at least 24 hours in order to block theremaining bind sites of the plate.

50 μl of Buffer A (0.1% BSA, 0.1M NaCl, 1 mM MgCl₂, 0.05% CHAPS and 0.1%NaN₃ in 20 mM phosphate buffer, pH 7.0) and 100 μl of mouse anti-partialpeptide of PACAP receptor-antiserum diluted with Buffer A were added toeach of the well of the above anti-mouse immunoglobulin bound microplateand to react at 4° C. for 16 hours. After the plate wase washed withPBS, 100 μl of HRP-labelled peptide diluted to 300 times with Buffer B(0.1% BSA, 0.4M NaCl and 2 mM EDTA in 20 mM phosphate buffer, pH 7.0)was added to react at 4° C. for 7 hours. Then, the plate was washed withPBS and 100 μl of TMB microwell peroxidase substrate system (Kirkegaard& Perry Lab, Inc.) was added to each well to react them at roomtemperature for 10 minutes. 100 μl of 1M phosphoric acid was added toeach well to stop the reaction and their absorptions at 450 nm wasassayed with a plate reader (MTP-120, Corona).

(6) Preparation of Anti-partial Peptide of PACAP Receptor MonoclonalAntibody

On mice which show relatively high antibody value, final immunization byintravenous injection of 200 to 300 μg of immunogen in 0.25 to 0.3 ml ofphysiological saline was conducted. Spleens were enucleated from themice after 3 to 4 days of the final immunization and pressed andfiltered through a stainless mesh and the filtrate was suspended inEagle's Minimum Essential Medium (MEM) to obtain a spleen cellsuspension. Mieloma cell P3-X63.Ag8.U1(P3U1 cell) derived from BALB/cmouse was used as a cell for cell fusion Current Topics in Microbiologyand Immunology, 81, 1(1978)!. The cell fusion was conducted according tothe original method. Spleen cells and P3U1 cells were respectivelywashed 3 times with MEM having no serum, and then they were mixed at 5:1in the ratio of spleen cells to P3U1 cells followed by centrifugation at700 rpm for 15 minutes to make the cells precipitate. After thoroughlyremoving the supernatant, the precipitate was softly mixed and 0.4 ml of45% polyethyleneglicol (PEG) 6000 (Kochlight) was added thereto and themixture was maintained in water bath at 37° C. for 7 minutes for thehybridization. 15 ml of MEM was slowly added by 2 ml per minute theretoand the mixture was centrifuged at 750 rpm for 15 minutes to obtain thecell precipitate. The cells were suspended mildly into 200 ml of GITmedium containing 10% fetal calf serum (Wako Pure Chemical Industry,Japan) (GIT-10% FCS) and a 24 well multidish(Limbro) was seeded with 1ml of the suspension to each well and incubated in an incubator with 5%carbonic acid at 37° C. After 24 hours of the incubation, 1 ml ofGIT-10% FCS containing HAT (0.1 mM hypoxanthine, 0.4 μM aminopterin, 1.6mM thymidine) (HAT medium) was added to each well and HAT selectivecultivation began. After 4 and 8 days from the beginning of thecultivation, 1 ml of the culture solution was changed with new HATmedium. Growth of hybridoma was found after 8 to 10 days from the cellfusion and the supernatant when the culture solution changed yellow wastaken and assayed according to the method described in Example 5.

Typical screening of hybridoma derived from mice immunized with apartial peptide of PACAP receptoris shown in FIG. 52. There are a fewcolonies of hybridoma in the wells and 3 wells were chosen, cloning ofan antibody producing strain with limiting dilution analysis wasconducted to obtain three hybridomas which produce anti-partial peptideof PACAP receptor (PRN1-25, PRN1-109 and PRN1-159). As a feeder cell forthe cloning, thymus cells of BALB/c mouse was employed. One(1) tothree(3)×10⁶ cells of these hybridomas were intraabdominallyadministered to BALB/c mice to which 0.5 ml of mineral oil wasintrabdominally administered, and 10 to 15 days after theadministration, ascites containing antibodies was collected.

Monoclonal antibodies were purified from the obtained ascites using acolumn to which Protein A was fixed. Thus, the ascites was diluted withequivalent binding buffer (3.5M NaCl, 0.05% NaN₃ in 1.5M glycine, pH9.0) and the dilution was applied on Recombinant Protein A-agarose(Repligen) equilibrized with the binding buffer, washed with the bufferand antibodies were eluted with an elution buffer (0.1M citrate bufferwith 0.05% NaN₃, pH 3.0). The purified monoclonal antibodies eluted weredialyzed against PBS containing 0.05% NaN₃ at 4° C. for two days and thedialysate was restored at 4° C. The monoclonal antibodies obtained areshown in Table 4.

                  TABLE 4    ______________________________________    Monoclonal antibodies                      Type    ______________________________________    PRN1-25a          IgG1    PRN1-109a         IgG1    PRN1-159a         IgG1    ______________________________________

(7) Detection of PACAP Receptor by Western Blotting with Anti-partialPeptide of PACAP Receptor Antibody

Human PACAP receptor was expressed in an insect cell using Baculo virusand a membrane fraction was prepared from the cell. Membrane protein wassolubilized with digitonin from the membrane fraction and concentratedon DEAE-Toyopearl column. The concentrated membrane protein solution wasisolated with SDS-polyacrylamide electrophoresis and transfered to PVDFmembrane (Applied Biosystem). The PVDF membrane transfered with proteinwas immersed in 5% BSA solution at 37° C. for 1 hour to saturateadsorption sites. The PVDF membrane was washed and immersed in 10 μg/mlPRN1-159a antibody solution at room temperature for 1 hour. Afterwashing, the membrane was immersed at room temperature for 2 hour in asolution with golden-colloid-labelled anti-mouse IgG and anti-mouse IgMantibodies (Amershum, Auroprobe BL plus GAM IgG+IgM). After washing, themembrane was treated with a sensitizer (Amershum, Intense BL silverenhancement Kit) and a band of PACAP receptor which was recognized withthe antibodies was detected (FIG. 53).

(8) Inhibition of PACAP binding by monoclonal antibodies

Membrane protein was solubilized with digitonin from the bovine brainmembrane fraction and concentrated on DEAE-Toyopearl column. Theconcentrated membrane protein solution was diluted with assay buffer (20mM Tris, 5 mM magnesium acetate, 2mM EGTA, 0.1% BSA, 0.05% digitonin,0.03% NaN₃, 0.5 mM PMSF, 20 μg/ml leupeptin, 4 μg/ml E-64, 1 μg/mlpepstatin, pH 7.2) to 90 μl and was added with 10 μl of the purifiedmonoclonal antibody solution. After mixing, the solution was kept at 4°C. for 16 hours and 2 μl of 5 nM radioactive iodine-labelled PACAP27 (¹²⁵ I!PACAP27) solution was added thereto to react at 25° C. for 1 hour.After completion of incubation, 1.5 ml of assay buffer detergent(digitonin in assay buffer was substituted with CHAPS of equivalentconcentration) was added to the reaction solution and then the solutionwas filtered on a glass-fiber filter paper which was previously treatedwith 0.3% polyethyleneimine. The filter paper was further washed withequivalent amount of assay buffer detergent and the capturedradio-activity was counted and radio-active PACAP27 bound to thereceptor was determined. As shown in FIG. 54, PRN1-159a inhibitedbinding of ¹²⁵ I!PACAP27 to the receptor.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled andpurview of this Application and the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 56    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    MetHisSerAspCysIlePheLysLysGluGlnAlaMetCysLeuGlu    151015    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    SerSerProGlyCysProGlyMetTrpAspAsnIleThrCysTrpLys    151015    ProAla    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ValGlyGluMetValLeuValSerCysProGlu    1510    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    PheArgIlePheAsnProAspGlnValTrpGluThrGluThrIleGly    151015    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    ArgAsnCysThrGluAspGlyTrpSerGluProPheProHisTyrPhe    151015    AspAlaCysGlyPhe    20    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    AspGlnAspTyrTyrTyrLeuSerValLysAlaLeuTyrThrValGly    151015    TyrSerThrSerLeu    20    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 50 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    ThrLeuThrThrAlaMetValIleLeuCysArgPheArgLysLeuHis    151015    CysThrArgAsnPheIleHisMetAsnLeuPheValSerPheMetLeu    202530    ArgAlaIleSerValPheIleLysAspTrpIleLeuTyrAlaGluGln    354045    AspSer    50    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 60 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    SerThrValGluCysLysAlaValMetValPhePheHisTyrCysVal    151015    ValSerAsnTyrPheTrpLeuPheIleGluGlyLeuTyrLeuPheThr    202530    LeuLeuValGluThrPhePheProGluArgArgTyrPheTyrTrpTyr    354045    ThrIleIleGlyTrpGlyThrProThrValCysVal    505560    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    ThrAlaLeuTrpTrpValIleLysGlyProValValGlySerIleMet    151015    ValAsnPheValLeuPheIleGlyIleIle    2025    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    IleLeuValGlnLysLeuGlnSerProAspMetGlyGlyAsnGluSer    151015    SerIleTyr    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 76 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    LeuArgLeuAlaArgSerThrLeuLeuLeuIleProLeuPheGlyIle    151015    HisTyrThrValPheAlaPheSerProGluAsnValSerLysArgGlu    202530    ArgLeuValPheGluLeuGlyLeuGlySerPheGlnGlyPheValVal    354045    AlaValLeuTyrCysPheLeuAsnGlyGluValGlnAlaGluIleLys    505560    ArgLysTrpArgSerTrpLysValAsnArgTyrPhe    657075    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    AspPheLysHisArgHisProSerLeuAlaSerSerGlyValAsnGly    151015    GlyThrGlnLeuSerIleLeuSerLysSerSerSerGlnIleArgMet    202530    Ser    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    MetHisSerAspCysIlePheLysLysGluGlnAlaMetCysLeuGlu    151015    LysIleGlnArgValAsnAspLeuMetGlyLeuAsnAsp    2025    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 476 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    MetHisSerAspCysIlePheLysLysGluGlnAlaMetCysLeuGlu    151015    LysIleGlnArgValAsnAspLeuMetGlyLeuAsnAspSerSerPro    202530    GlyCysProGlyMetTrpAspAsnIleThrCysTrpLysProAlaHis    354045    ValGlyGluMetValLeuValSerCysProGluLeuPheArgIlePhe    505560    AsnProAspGlnValTrpGluThrGluThrIleGlyGluPheGlyPhe    65707580    AlaAspSerLysSerLeuAspLeuSerAspMetArgValValSerArg    859095    AsnCysThrGluAspGlyTrpSerGluProPheProHisTyrPheAsp    100105110    AlaCysGlyPheGluGluTyrGluSerGluThrGlyAspGlnAspTyr    115120125    TyrTyrLeuSerValLysAlaLeuTyrThrValGlyTyrSerThrSer    130135140    LeuValThrLeuThrThrAlaMetValIleLeuCysArgPheArgLys    145150155160    LeuHisCysThrArgAsnPheIleHisMetAsnLeuPheValSerPhe    165170175    MetLeuArgAlaIleSerValPheIleLysAspTrpIleLeuTyrAla    180185190    GluGlnAspSerAsnHisCysPheValSerThrValGluCysLysAla    195200205    ValMetValPhePheHisTyrCysValValSerAsnTyrPheTrpLeu    210215220    PheIleGluGlyLeuTyrLeuPheThrLeuLeuValGluThrPhePhe    225230235240    ProGluArgArgTyrPheTyrTrpTyrIleIleIleGlyTrpGlyThr    245250255    ProThrValCysValSerValTrpAlaMetLeuArgLeuTyrPheAsp    260265270    AspThrGlyCysTrpAspMetAsnAspAsnThrAlaLeuTrpTrpVal    275280285    IleLysGlyProValValGlySerIleMetValAsnPheValLeuPhe    290295300    IleGlyIleIleValIleLeuValGlnLysLeuGlnSerProAspMet    305310315320    GlyGlyAsnGluSerSerIleTyrPheSerCysValGlnLysCysTyr    325330335    CysLysProGlnArgAlaGlnGlnHisSerCysLysMetSerGluLeu    340345350    SerThrIleThrLeuArgLeuAlaArgSerThrLeuLeuLeuIlePro    355360365    LeuPheGlyIleHisTyrThrValPheAlaPheSerProGluAsnVal    370375380    SerLysArgGluArgLeuValPheGluLeuGlyLeuGlySerPheGln    385390395400    GlyPheValValAlaValLeuTyrCysPheLeuAsnGlyGluValGln    405410415    AlaGluIleLysArgLysTrpArgSerTrpLysValAsnArgTyrPhe    420425430    ThrMetAspPheLysHisArgHisProSerLeuAlaSerSerGlyVal    435440445    AsnGlyGlyThrGlnLeuSerIleLeuSerLysSerSerSerGlnIle    450455460    ArgMetSerGlyLeuProAlaAspAsnLeuAlaThr    465470475    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 513 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    MetArgGlyGlyArgHisTrpProGluProProCysArgLeuArgSer    151015    ValMetAlaSerIleAlaGlnValSerLeuAlaAlaLeuLeuLeuLeu    202530    ProMetAlaThrAlaMetHisSerAspCysIlePheLysLysGluGln    354045    AlaMetCysLeuGluLysIleGlnArgValAsnAspLeuMetGlyLeu    505560    AsnAspSerSerProGlyCysProGlyMetTrpAspAsnIleThrCys    65707580    TrpLysProAlaHisValGlyGluMetValLeuValSerCysProGlu    859095    LeuPheArgIlePheAsnProAspGlnValTrpGluThrGluThrIle    100105110    GlyGluPheGlyPheAlaAspSerLysSerLeuAspLeuSerAspMet    115120125    ArgValValSerArgAsnCysThrGluAspGlyTrpSerGluProPhe    130135140    ProHisTyrPheAspAlaCysGlyPheGluGluTyrGluSerGluThr    145150155160    GlyAspGlnAspTyrTyrTyrLeuSerValLysAlaLeuTyrThrVal    165170175    GlyTyrSerThrSerLeuValThrLeuThrThrAlaMetValIleLeu    180185190    CysArgPheArgLysLeuHisCysThrArgAsnPheIleHisMetAsn    195200205    LeuPheValSerPheMetLeuArgAlaIleSerValPheIleLysAsp    210215220    TrpIleLeuTyrAlaGluGlnAspSerAsnHisCysPheValSerThr    225230235240    ValGluCysLysAlaValMetValPhePheHisTyrCysValValSer    245250255    AsnTyrPheTrpLeuPheIleGluGlyLeuTyrLeuPheThrLeuLeu    260265270    ValGluThrPhePheProGluArgArgTyrPheTyrTrpTyrIleIle    275280285    IleGlyTrpGlyThrProThrValCysValSerValTrpAlaMetLeu    290295300    ArgLeuTyrPheAspAspThrGlyCysTrpAspMetAsnAspAsnThr    305310315320    AlaLeuTrpTrpValIleLysGlyProValValGlySerIleMetVal    325330335    AsnPheValLeuPheIleGlyIleIleValIleLeuValGlnLysLeu    340345350    GlnSerProAspMetGlyGlyAsnGluSerSerIleTyrPheSerCys    355360365    ValGlnLysCysTyrCysLysProGlnArgAlaGlnGlnHisSerCys    370375380    LysMetSerGluLeuSerThrIleThrLeuArgLeuAlaArgSerThr    385390395400    LeuLeuLeuIleProLeuPheGlyIleHisTyrThrValPheAlaPhe    405410415    SerProGluAsnValSerLysArgGluArgLeuValPheGluLeuGly    420425430    LeuGlySerPheGlnGlyPheValValAlaValLeuTyrCysPheLeu    435440445    AsnGlyGluValGlnAlaGluIleLysArgLysTrpArgSerTrpLys    450455460    ValAsnArgTyrPheThrMetAspPheLysHisArgHisProSerLeu    465470475480    AlaSerSerGlyValAsnGlyGlyThrGlnLeuSerIleLeuSerLys    485490495    SerSerSerGlnIleArgMetSerGlyLeuProAlaAspAsnLeuAla    500505510    Thr    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 448 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    MetHisSerAspCysIlePheLysLysGluGlnAlaMetCysLeuGlu    151015    LysIleGlnArgValAsnAspLeuMetGlyLeuAsnAspSerSerPro    202530    GlyCysProGlyMetTrpAspAsnIleThrCysTrpLysProAlaHis    354045    ValGlyGluMetValLeuValSerCysProGluLeuPheArgIlePhe    505560    AsnProAspGlnValTrpGluThrGluThrIleGlyGluPheGlyPhe    65707580    AlaAspSerLysSerLeuAspLeuSerAspMetArgValValSerArg    859095    AsnCysThrGluAspGlyTrpSerGluProPheProHisTyrPheAsp    100105110    AlaCysGlyPheGluGluTyrGluSerGluThrGlyAspGlnAspTyr    115120125    TyrTyrLeuSerValLysAlaLeuTyrThrValGlyTyrSerThrSer    130135140    LeuValThrLeuThrThrAlaMetValIleLeuCysArgPheArgLys    145150155160    LeuHisCysThrArgAsnPheIleHisMetAsnLeuPheValSerPhe    165170175    MetLeuArgAlaIleSerValPheIleLysAspTrpIleLeuTyrAla    180185190    GluGlnAspSerAsnHisCysPheValSerThrValGluCysLysAla    195200205    ValMetValPhePheHisTyrCysValValSerAsnTyrPheTrpLeu    210215220    PheIleGluGlyLeuTyrLeuPheThrLeuLeuValGluThrPhePhe    225230235240    ProGluArgArgTyrPheTyrTrpTyrIleIleIleGlyTrpGlyThr    245250255    ProThrValCysValSerValTrpAlaMetLeuArgLeuTyrPheAsp    260265270    AspThrGlyCysTrpAspMetAsnAspAsnThrAlaLeuTrpTrpVal    275280285    IleLysGlyProValValGlySerIleMetValAsnPheValLeuPhe    290295300    IleGlyIleIleValIleLeuValGlnLysLeuGlnSerProAspMet    305310315320    GlyGlyAsnGluSerSerIleTyrLeuArgLeuAlaArgSerThrLeu    325330335    LeuLeuIleProLeuPheGlyIleHisTyrThrValPheAlaPheSer    340345350    ProGluAsnValSerLysArgGluArgLeuValPheGluLeuGlyLeu    355360365    GlySerPheGlnGlyPheValValAlaValLeuTyrCysPheLeuAsn    370375380    GlyGluValGlnAlaGluIleLysArgLysTrpArgSerTrpLysVal    385390395400    AsnArgTyrPheThrMetAspPheLysHisArgHisProSerLeuAla    405410415    SerSerGlyValAsnGlyGlyThrGlnLeuSerIleLeuSerLysSer    420425430    SerSerGlnIleArgMetSerGlyLeuProAlaAspAsnLeuAlaThr    435440445    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 485 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    MetArgGlyGlyArgHisTrpProGluProProCysArgLeuArgSer    151015    ValMetAlaSerIleAlaGlnValSerLeuAlaAlaLeuLeuLeuLeu    202530    ProMetAlaThrAlaMetHisSerAspCysIlePheLysLysGluGln    354045    AlaMetCysLeuGluLysIleGlnArgValAsnAspLeuMetGlyLeu    505560    AsnAspSerSerProGlyCysProGlyMetTrpAspAsnIleThrCys    65707580    TrpLysProAlaHisValGlyGluMetValLeuValSerCysProGlu    859095    LeuPheArgIlePheAsnProAspGlnValTrpGluThrGluThrIle    100105110    GlyGluPheGlyPheAlaAspSerLysSerLeuAspLeuSerAspMet    115120125    ArgValValSerArgAsnCysThrGluAspGlyTrpSerGluProPhe    130135140    ProHisTyrPheAspAlaCysGlyPheGluGluTyrGluSerGluThr    145150155160    GlyAspGlnAspTyrTyrTyrLeuSerValLysAlaLeuTyrThrVal    165170175    GlyTyrSerThrSerLeuValThrLeuThrThrAlaMetValIleLeu    180185190    CysArgPheArgLysLeuHisCysThrArgAsnPheIleHisMetAsn    195200205    LeuPheValSerPheMetLeuArgAlaIleSerValPheIleLysAsp    210215220    TrpIleLeuTyrAlaGluGlnAspSerAsnHisCysPheValSerThr    225230235240    ValGluCysLysAlaValMetValPhePheHisTyrCysValValSer    245250255    AsnTyrPheTrpLeuPheIleGluGlyLeuTyrLeuPheThrLeuLeu    260265270    ValGluThrPhePheProGluArgArgTyrPheTyrTrpTyrIleIle    275280285    IleGlyTrpGlyThrProThrValCysValSerValTrpAlaMetLeu    290295300    ArgLeuTyrPheAspAspThrGlyCysTrpAspMetAsnAspAsnThr    305310315320    AlaLeuTrpTrpValIleLysGlyProValValGlySerIleMetVal    325330335    AsnPheValLeuPheIleGlyIleIleValIleLeuValGlnLysLeu    340345350    GlnSerProAspMetGlyGlyAsnGluSerSerIleTyrLeuArgLeu    355360365    AlaArgSerThrLeuLeuLeuIleProLeuPheGlyIleHisTyrThr    370375380    ValPheAlaPheSerProGluAsnValSerLysArgGluArgLeuVal    385390395400    PheGluLeuGlyLeuGlySerPheGlnGlyPheValValAlaValLeu    405410415    TyrCysPheLeuAsnGlyGluValGlnAlaGluIleLysArgLysTrp    420425430    ArgSerTrpLysValAsnArgTyrPheThrMetAspPheLysHisArg    435440445    HisProSerLeuAlaSerSerGlyValAsnGlyGlyThrGlnLeuSer    450455460    IleLeuSerLysSerSerSerGlnIleArgMetSerGlyLeuProAla    465470475480    AspAsnLeuAlaThr    485    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 448 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    MetHisSerAspCysIlePheLysLysGluGlnAlaMetCysLeuGlu    151015    ArgIleGlnArgAlaAsnAspLeuMetGlyLeuAsnGluSerSerPro    202530    GlyCysProGlyMetTrpAspAsnIleThrCysTrpLysProAlaGln    354045    ValGlyGluMetValLeuValSerCysProGluValPheArgIlePhe    505560    AsnProAspGlnValTrpMetThrGluThrIleGlyAspSerGlyPhe    65707580    AlaAspSerAsnSerLeuGluIleThrAspMetGlyValValGlyArg    859095    AsnCysThrGluAspGlyTrpSerGluProPheProHisTyrPheAsp    100105110    AlaCysGlyPheAspAspTyrGluProGluSerGlyAspGlnAspTyr    115120125    TyrTyrLeuSerValLysAlaLeuTyrThrValGlyTyrSerThrSer    130135140    LeuAlaThrLeuThrThrAlaMetValIleLeuCysArgPheArgLys    145150155160    LeuHisCysThrArgAsnPheIleHisMetAsnLeuPheValSerPhe    165170175    MetLeuArgAlaIleSerValPheIleLysAspTrpIleLeuTyrAla    180185190    GluGlnAspSerSerHisCysPheValSerThrValGluCysLysAla    195200205    ValMetValPhePheHisTyrCysValValSerAsnTyrPheTrpLeu    210215220    PheIleGluGlyLeuTyrLeuPheThrLeuLeuValGluThrPhePhe    225230235240    ProGluArgArgTyrPheTyrTrpTyrThrIleIleGlyTrpGlyThr    245250255    ProThrValCysValThrValTrpAlaValLeuArgLeuTyrPheAsp    260265270    AspAlaGlyCysTrpAspMetAsnAspSerThrAlaLeuTrpTrpVal    275280285    IleLysGlyProValValGlySerIleMetValAsnPheValLeuPhe    290295300    IleGlyIleIleIleIleLeuValGlnLysLeuGlnSerProAspMet    305310315320    GlyGlyAsnGluSerSerIleTyrLeuArgLeuAlaArgSerThrLeu    325330335    LeuLeuIleProLeuPheGlyIleHisTyrThrValPheAlaPheSer    340345350    ProGluAsnValSerLysArgGluArgLeuValPheGluLeuGlyLeu    355360365    GlySerPheGlnGlyPheValValAlaValLeuTyrCysPheLeuAsn    370375380    GlyGluValGlnAlaGluIleLysArgLysTrpArgSerTrpLysVal    385390395400    AsnArgTyrPheThrMetAspPheLysHisArgHisProSerLeuAla    405410415    SerSerGlyValAsnGlyGlyThrGlnLeuSerIleLeuSerLysSer    420425430    SerSerGlnLeuArgMetSerSerLeuProAlaAspAsnLeuAlaThr    435440445    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 467 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    MetAlaArgValLeuGlnLeuSerLeuThrAlaLeuLeuLeuProVal    151015    AlaIleAlaMetHisSerAspCysIlePheLysLysGluGlnAlaMet    202530    CysLeuGluArgIleGlnArgAlaAsnAspLeuMetGlyLeuAsnGlu    354045    SerSerProGlyCysProGlyMetTrpAspAsnIleThrCysTrpLys    505560    ProAlaGlnValGlyGluMetValLeuValSerCysProGluValPhe    65707580    ArgIlePheAsnProAspGlnValTrpMetThrGluThrIleGlyAsp    859095    SerGlyPheAlaAspSerAsnSerLeuGluIleThrAspMetGlyVal    100105110    ValGlyArgAsnCysThrGluAspGlyTrpSerGluProPheProHis    115120125    TyrPheAspAlaCysGlyPheAspAspTyrGluProGluSerGlyAsp    130135140    GlnAspTyrTyrTyrLeuSerValLysAlaLeuTyrThrValGlyTyr    145150155160    SerThrSerLeuAlaThrLeuThrThrAlaMetValIleLeuCysArg    165170175    PheArgLysLeuHisCysThrArgAsnPheIleHisMetAsnLeuPhe    180185190    ValSerPheMetLeuArgAlaIleSerValPheIleLysAspTrpIle    195200205    LeuTyrAlaGluGlnAspSerSerHisCysPheValSerThrValGlu    210215220    CysLysAlaValMetValPhePheHisTyrCysValValSerAsnTyr    225230235240    PheTrpLeuPheIleGluGlyLeuTyrLeuPheThrLeuLeuValGlu    245250255    ThrPhePheProGluArgArgTyrPheTyrTrpTyrThrIleIleGly    260265270    TrpGlyThrProThrValCysValThrValTrpAlaValLeuArgLeu    275280285    TyrPheAspAspAlaGlyCysTrpAspMetAsnAspSerThrAlaLeu    290295300    TrpTrpValIleLysGlyProValValGlySerIleMetValAsnPhe    305310315320    ValLeuPheIleGlyIleIleIleIleLeuValGlnLysLeuGlnSer    325330335    ProAspMetGlyGlyAsnGluSerSerIleTyrLeuArgLeuAlaArg    340345350    SerThrLeuLeuLeuIleProLeuPheGlyIleHisTyrThrValPhe    355360365    AlaPheSerProGluAsnValSerLysArgGluArgLeuValPheGlu    370375380    LeuGlyLeuGlySerPheGlnGlyPheValValAlaValLeuTyrCys    385390395400    PheLeuAsnGlyGluValGlnAlaGluIleLysArgLysTrpArgSer    405410415    TrpLysValAsnArgTyrPheThrMetAspPheLysHisArgHisPro    420425430    SerLeuAlaSerSerGlyValAsnGlyGlyThrGlnLeuSerIleLeu    435440445    SerLysSerSerSerGlnLeuArgMetSerSerLeuProAlaAspAsn    450455460    LeuAlaThr    465    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 476 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    MetHisSerAspCysIlePheLysLysGluGlnAlaMetCysLeuGlu    151015    ArgIleGlnArgAlaAsnAspLeuMetGlyLeuAsnGluSerSerPro    202530    GlyCysProGlyMetTrpAspAsnIleThrCysTrpLysProAlaGln    354045    ValGlyGluMetValLeuValSerCysProGluValPheArgIlePhe    505560    AsnProAspGlnValTrpMetThrGluThrIleGlyAspSerGlyPhe    65707580    AlaAspSerAsnSerLeuGluIleThrAspMetGlyValValGlyArg    859095    AsnCysThrGluAspGlyTrpSerGluProPheProHisTyrPheAsp    100105110    AlaCysGlyPheAspAspTyrGluProGluSerGlyAspGlnAspTyr    115120125    TyrTyrLeuSerValLysAlaLeuTyrThrValGlyTyrSerThrSer    130135140    LeuAlaThrLeuThrThrAlaMetValIleLeuCysArgPheArgLys    145150155160    LeuHisCysThrArgAsnPheIleHisMetAsnLeuPheValSerPhe    165170175    MetLeuArgAlaIleSerValPheIleLysAspTrpIleLeuTyrAla    180185190    GluGlnAspSerSerHisCysPheValSerThrValGluCysLysAla    195200205    ValMetValPhePheHisTyrCysValValSerAsnTyrPheTrpLeu    210215220    PheIleGluGlyLeuTyrLeuPheThrLeuLeuValGluThrPhePhe    225230235240    ProGluArgArgTyrPheTyrTrpTyrThrIleIleGlyTrpGlyThr    245250255    ProThrValCysValThrValTrpAlaValLeuArgLeuTyrPheAsp    260265270    AspAlaGlyCysTrpAspMetAsnAspSerThrAlaLeuTrpTrpVal    275280285    IleLysGlyProValValGlySerIleMetValAsnPheValLeuPhe    290295300    IleGlyIleIleIleIleLeuValGlnLysLeuGlnSerProAspMet    305310315320    GlyGlyAsnGluSerSerIleTyrPheSerCysValGlnLysCysTyr    325330335    CysLysProGlnArgAlaGlnGlnHisSerCysLysMetSerGluLeu    340345350    SerThrIleThrLeuArgLeuAlaArgSerThrLeuLeuLeuIlePro    355360365    LeuPheGlyIleHisTyrThrValPheAlaPheSerProGluAsnVal    370375380    SerLysArgGluArgLeuValPheGluLeuGlyLeuGlySerPheGln    385390395400    GlyPheValValAlaValLeuTyrCysPheLeuAsnGlyGluValGln    405410415    AlaGluIleLysArgLysTrpArgSerTrpLysValAsnArgTyrPhe    420425430    ThrMetAspPheLysHisArgHisProSerLeuAlaSerSerGlyVal    435440445    AsnGlyGlyThrGlnLeuSerIleLeuSerLysSerSerSerGlnLeu    450455460    ArgMetSerSerLeuProAlaAspAsnLeuAlaThr    465470475    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 495 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    MetAlaArgValLeuGlnLeuSerLeuThrAlaLeuLeuLeuProVal    151015    AlaIleAlaMetHisSerAspCysIlePheLysLysGluGlnAlaMet    202530    CysLeuGluArgIleGlnArgAlaAsnAspLeuMetGlyLeuAsnGlu    354045    SerSerProGlyCysProGlyMetTrpAspAsnIleThrCysTrpLys    505560    ProAlaGlnValGlyGluMetValLeuValSerCysProGluValPhe    65707580    ArgIlePheAsnProAspGlnValTrpMetThrGluThrIleGlyAsp    859095    SerGlyPheAlaAspSerAsnSerLeuGluIleThrAspMetGlyVal    100105110    ValGlyArgAsnCysThrGluAspGlyTrpSerGluProPheProHis    115120125    TyrPheAspAlaCysGlyPheAspAspTyrGluProGluSerGlyAsp    130135140    GlnAspTyrTyrTyrLeuSerValLysAlaLeuTyrThrValGlyTyr    145150155160    SerThrSerLeuAlaThrLeuThrThrAlaMetValIleLeuCysArg    165170175    PheArgLysLeuHisCysThrArgAsnPheIleHisMetAsnLeuPhe    180185190    ValSerPheMetLeuArgAlaIleSerValPheIleLysAspTrpIle    195200205    LeuTyrAlaGluGlnAspSerSerHisCysPheValSerThrValGlu    210215220    CysLysAlaValMetValPhePheHisTyrCysValValSerAsnTyr    225230235240    PheTrpLeuPheIleGluGlyLeuTyrLeuPheThrLeuLeuValGlu    245250255    ThrPhePheProGluArgArgTyrPheTyrTrpTyrThrIleIleGly    260265270    TrpGlyThrProThrValCysValThrValTrpAlaValLeuArgLeu    275280285    TyrPheAspAspAlaGlyCysTrpAspMetAsnAspSerThrAlaLeu    290295300    TrpTrpValIleLysGlyProValValGlySerIleMetValAsnPhe    305310315320    ValLeuPheIleGlyIleIleIleIleLeuValGlnLysLeuGlnSer    325330335    ProAspMetGlyGlyAsnGluSerSerIleTyrPheSerCysValGln    340345350    LysCysTyrCysLysProGlnArgAlaGlnGlnHisSerCysLysMet    355360365    SerGluLeuSerThrIleThrLeuArgLeuAlaArgSerThrLeuLeu    370375380    LeuIleProLeuPheGlyIleHisTyrThrValPheAlaPheSerPro    385390395400    GluAsnValSerLysArgGluArgLeuValPheGluLeuGlyLeuGly    405410415    SerPheGlnGlyPheValValAlaValLeuTyrCysPheLeuAsnGly    420425430    GluValGlnAlaGluIleLysArgLysTrpArgSerTrpLysValAsn    435440445    ArgTyrPheThrMetAspPheLysHisArgHisProSerLeuAlaSer    450455460    SerGlyValAsnGlyGlyThrGlnLeuSerIleLeuSerLysSerSer    465470475480    SerGlnLeuArgMetSerSerLeuProAlaAspAsnLeuAlaThr    485490495    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 448 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    MetHisSerAspCysIlePheLysLysGluGlnAlaMetCysLeuGlu    151015    LysIleGlnArgAlaAsnGluLeuMetGlyPheAsnAspSerSerPro    202530    GlyCysProGlyMetTrpAspAsnIleThrCysTrpLysProAlaHis    354045    ValGlyGluMetValLeuValSerCysProGluLeuPheArgIlePhe    505560    AsnProAspGlnValTrpGluThrGluThrIleGlyGluSerAspPhe    65707580    GlyAspSerAsnSerLeuAspLeuSerAspMetGlyValValSerArg    859095    AsnCysThrGluAspGlyTrpSerGluProPheProHisTyrPheAsp    100105110    AlaCysGlyPheAspGluTyrGluSerGluThrGlyAspGlnAspTyr    115120125    TyrTyrLeuSerValLysAlaLeuTyrThrValGlyTyrSerThrSer    130135140    LeuValThrLeuThrThrAlaMetValIleLeuCysArgPheArgLys    145150155160    LeuHisCysThrArgAsnPheIleHisMetAsnLeuPheValSerPhe    165170175    MetLeuArgAlaIleSerValPheIleLysAspTrpIleLeuTyrAla    180185190    GluGlnAspSerAsnHisCysPheIleSerThrValGluCysLysAla    195200205    ValMetValPhePheHisTyrCysValValSerAsnTyrPheTrpLeu    210215220    PheIleGluGlyLeuTyrLeuPheThrLeuLeuValGluThrPhePhe    225230235240    ProGluArgArgTyrPheTyrTrpTyrThrIleIleGlyTrpGlyThr    245250255    ProThrValCysValThrValTrpAlaThrLeuArgLeuTyrPheAsp    260265270    AspThrGlyCysTrpAspMetAsnAspSerThrAlaLeuTrpTrpVal    275280285    IleLysGlyProValValGlySerIleMetValAsnPheValLeuPhe    290295300    IleGlyIleIleValIleLeuValGlnLysLeuGlnSerProAspMet    305310315320    GlyGlyAsnGluSerSerIleTyrLeuArgLeuAlaArgSerThrLeu    325330335    LeuLeuIleProLeuPheGlyIleHisTyrThrValPheAlaPheSer    340345350    ProGluAsnValSerLysArgGluArgLeuValPheGluLeuGlyLeu    355360365    GlySerPheGlnGlyPheValValAlaValLeuTyrCysPheLeuAsn    370375380    GlyGluValGlnAlaGluIleLysArgLysTrpArgSerTrpLysVal    385390395400    AsnArgTyrPheAlaValAspPheLysHisArgHisProSerLeuAla    405410415    SerSerGlyValAsnGlyGlyThrGlnLeuSerIleLeuSerLysSer    420425430    SerSerGlnIleArgMetSerGlyLeuProAlaAspAsnLeuAlaThr    435440445    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 525 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    MetAlaGlyValValHisValSerLeuAlaAlaHisCysGlyAlaCys    151015    ProTrpGlyArgGlyArgLeuArgLysGlyArgAlaAlaCysLysSer    202530    AlaAlaGlnArgHisIleGlyAlaAspLeuProLeuLeuSerValGly    354045    GlyGlnTrpCysTrpProArgSerValMetAlaGlyValValHisVal    505560    SerLeuAlaAlaLeuLeuLeuLeuProMetAlaProAlaMetHisSer    65707580    AspCysIlePheLysLysGluGlnAlaMetCysLeuGluLysIleGln    859095    ArgAlaAsnGluLeuMetGlyPheAsnAspSerSerProGlyCysPro    100105110    GlyMetTrpAspAsnIleThrCysTrpLysProAlaHisValGlyGlu    115120125    MetValLeuValSerCysProGluLeuPheArgIlePheAsnProAsp    130135140    GlnValTrpGluThrGluThrIleGlyGluSerAspPheGlyAspSer    145150155160    AsnSerLeuAspLeuSerAspMetGlyValValSerArgAsnCysThr    165170175    GluAspGlyTrpSerGluProPheProHisTyrPheAspAlaCysGly    180185190    PheAspGluTyrGluSerGluThrGlyAspGlnAspTyrTyrTyrLeu    195200205    SerValLysAlaLeuTyrThrValGlyTyrSerThrSerLeuValThr    210215220    LeuThrThrAlaMetValIleLeuCysArgPheArgLysLeuHisCys    225230235240    ThrArgAsnPheIleHisMetAsnLeuPheValSerPheMetLeuArg    245250255    AlaIleSerValPheIleLysAspTrpIleLeuTyrAlaGluGlnAsp    260265270    SerAsnHisCysPheIleSerThrValGluCysLysAlaValMetVal    275280285    PhePheHisTyrCysValValSerAsnTyrPheTrpLeuPheIleGlu    290295300    GlyLeuTyrLeuPheThrLeuLeuValGluThrPhePheProGluArg    305310315320    ArgTyrPheTyrTrpTyrThrIleIleGlyTrpGlyThrProThrVal    325330335    CysValThrValTrpAlaThrLeuArgLeuTyrPheAspAspThrGly    340345350    CysTrpAspMetAsnAspSerThrAlaLeuTrpTrpValIleLysGly    355360365    ProValValGlySerIleMetValAsnPheValLeuPheIleGlyIle    370375380    IleValIleLeuValGlnLysLeuGlnSerProAspMetGlyGlyAsn    385390395400    GluSerSerIleTyrLeuArgLeuAlaArgSerThrLeuLeuLeuIle    405410415    ProLeuPheGlyIleHisTyrThrValPheAlaPheSerProGluAsn    420425430    ValSerLysArgGluArgLeuValPheGluLeuGlyLeuGlySerPhe    435440445    GlnGlyPheValValAlaValLeuTyrCysPheLeuAsnGlyGluVal    450455460    GlnAlaGluIleLysArgLysTrpArgSerTrpLysValAsnArgTyr    465470475480    PheAlaValAspPheLysHisArgHisProSerLeuAlaSerSerGly    485490495    ValAsnGlyGlyThrGlnLeuSerIleLeuSerLysSerSerSerGln    500505510    IleArgMetSerGlyLeuProAlaAspAsnLeuAlaThr    515520525    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 476 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    MetHisSerAspCysIlePheLysLysGluGlnAlaMetCysLeuGlu    151015    LysIleGlnArgAlaAsnGluLeuMetGlyPheAsnAspSerSerPro    202530    GlyCysProGlyMetTrpAspAsnIleThrCysTrpLysProAlaHis    354045    ValGlyGluMetValLeuValSerCysProGluLeuPheArgIlePhe    505560    AsnProAspGlnValTrpGluThrGluThrIleGlyGluSerAspPhe    65707580    GlyAspSerAsnSerLeuAspLeuSerAspMetGlyValValSerArg    859095    AsnCysThrGluAspGlyTrpSerGluProPheProHisTyrPheAsp    100105110    AlaCysGlyPheAspGluTyrGluSerGluThrGlyAspGlnAspTyr    115120125    TyrTyrLeuSerValLysAlaLeuTyrThrValGlyTyrSerThrSer    130135140    LeuValThrLeuThrThrAlaMetValIleLeuCysArgPheArgLys    145150155160    LeuHisCysThrArgAsnPheIleHisMetAsnLeuPheValSerPhe    165170175    MetLeuArgAlaIleSerValPheIleLysAspTrpIleLeuTyrAla    180185190    GluGlnAspSerAsnHisCysPheIleSerThrValGluCysLysAla    195200205    ValMetValPhePheHisTyrCysValValSerAsnTyrPheTrpLeu    210215220    PheIleGluGlyLeuTyrLeuPheThrLeuLeuValGluThrPhePhe    225230235240    ProGluArgArgTyrPheTyrTrpTyrThrIleIleGlyTrpGlyThr    245250255    ProThrValCysValThrValTrpAlaThrLeuArgLeuTyrPheAsp    260265270    AspThrGlyCysTrpAspMetAsnAspSerThrAlaLeuTrpTrpVal    275280285    IleLysGlyProValValGlySerIleMetValAsnPheValLeuPhe    290295300    IleGlyIleIleValIleLeuValGlnLysLeuGlnSerProAspMet    305310315320    GlyGlyAsnGluSerSerIleTyrPheSerCysValGlnLysCysTyr    325330335    CysLysProGlnArgAlaGlnGlnHisSerCysLysMetSerGluLeu    340345350    SerThrIleThrLeuArgLeuAlaArgSerThrLeuLeuLeuIlePro    355360365    LeuPheGlyIleHisTyrThrValPheAlaPheSerProGluAsnVal    370375380    SerLysArgGluArgLeuValPheGluLeuGlyLeuGlySerPheGln    385390395400    GlyPheValValAlaValLeuTyrCysPheLeuAsnGlyGluValGln    405410415    AlaGluIleLysArgLysTrpArgSerTrpLysValAsnArgTyrPhe    420425430    AlaValAspPheLysHisArgHisProSerLeuAlaSerSerGlyVal    435440445    AsnGlyGlyThrGlnLeuSerIleLeuSerLysSerSerSerGlnIle    450455460    ArgMetSerGlyLeuProAlaAspAsnLeuAlaThr    465470475    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 553 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    MetAlaGlyValValHisValSerLeuAlaAlaHisCysGlyAlaCys    151015    ProTrpGlyArgGlyArgLeuArgLysGlyArgAlaAlaCysLysSer    202530    AlaAlaGlnArgHisIleGlyAlaAspLeuProLeuLeuSerValGly    354045    GlyGlnTrpCysTrpProArgSerValMetAlaGlyValValHisVal    505560    SerLeuAlaAlaLeuLeuLeuLeuProMetAlaProAlaMetHisSer    65707580    AspCysIlePheLysLysGluGlnAlaMetCysLeuGluLysIleGln    859095    ArgAlaAsnGluLeuMetGlyPheAsnAspSerSerProGlyCysPro    100105110    GlyMetTrpAspAsnIleThrCysTrpLysProAlaHisValGlyGlu    115120125    MetValLeuValSerCysProGluLeuPheArgIlePheAsnProAsp    130135140    GlnValTrpGluThrGluThrIleGlyGluSerAspPheGlyAspSer    145150155160    AsnSerLeuAspLeuSerAspMetGlyValValSerArgAsnCysThr    165170175    GluAspGlyTrpSerGluProPheProHisTyrPheAspAlaCysGly    180185190    PheAspGluTyrGluSerGluThrGlyAspGlnAspTyrTyrTyrLeu    195200205    SerValLysAlaLeuTyrThrValGlyTyrSerThrSerLeuValThr    210215220    LeuThrThrAlaMetValIleLeuCysArgPheArgLysLeuHisCys    225230235240    ThrArgAsnPheIleHisMetAsnLeuPheValSerPheMetLeuArg    245250255    AlaIleSerValPheIleLysAspTrpIleLeuTyrAlaGluGlnAsp    260265270    SerAsnHisCysPheIleSerThrValGluCysLysAlaValMetVal    275280285    PhePheHisTyrCysValValSerAsnTyrPheTrpLeuPheIleGlu    290295300    GlyLeuTyrLeuPheThrLeuLeuValGluThrPhePheProGluArg    305310315320    ArgTyrPheTyrTrpTyrThrIleIleGlyTrpGlyThrProThrVal    325330335    CysValThrValTrpAlaThrLeuArgLeuTyrPheAspAspThrGly    340345350    CysTrpAspMetAsnAspSerThrAlaLeuTrpTrpValIleLysGly    355360365    ProValValGlySerIleMetValAsnPheValLeuPheIleGlyIle    370375380    IleValIleLeuValGlnLysLeuGlnSerProAspMetGlyGlyAsn    385390395400    GluSerSerIleTyrPheSerCysValGlnLysCysTyrCysLysPro    405410415    GlnArgAlaGlnGlnHisSerCysLysMetSerGluLeuSerThrIle    420425430    ThrLeuArgLeuAlaArgSerThrLeuLeuLeuIleProLeuPheGly    435440445    IleHisTyrThrValPheAlaPheSerProGluAsnValSerLysArg    450455460    GluArgLeuValPheGluLeuGlyLeuGlySerPheGlnGlyPheVal    465470475480    ValAlaValLeuTyrCysPheLeuAsnGlyGluValGlnAlaGluIle    485490495    LysArgLysTrpArgSerTrpLysValAsnArgTyrPheAlaValAsp    500505510    PheLysHisArgHisProSerLeuAlaSerSerGlyValAsnGlyGly    515520525    ThrGlnLeuSerIleLeuSerLysSerSerSerGlnIleArgMetSer    530535540    GlyLeuProAlaAspAsnLeuAlaThr    545550    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 475 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    MetHisSerAspCysIlePheLysLysGluGlnAlaMetCysLeuGlu    151015    LysIleGlnArgAlaAsnGluLeuMetGlyPheAsnAspSerSerPro    202530    GlyCysProGlyMetTrpAspAsnIleThrCysTrpLysProAlaHis    354045    ValGlyGluMetValLeuValSerCysProGluLeuPheArgIlePhe    505560    AsnProAspGlnValTrpGluThrGluThrIleGlyGluSerAspPhe    65707580    GlyAspSerAsnSerLeuAspLeuSerAspMetGlyValValSerArg    859095    AsnCysThrGluAspGlyTrpSerGluProPheProHisTyrPheAsp    100105110    AlaCysGlyPheAspGluTyrGluSerGluThrGlyAspGlnAspTyr    115120125    TyrTyrLeuSerValLysAlaLeuTyrThrValGlyTyrSerThrSer    130135140    LeuValThrLeuThrThrAlaMetValIleLeuCysArgPheArgLys    145150155160    LeuHisCysThrArgAsnPheIleHisMetAsnLeuPheValSerPhe    165170175    MetLeuArgAlaIleSerValPheIleLysAspTrpIleLeuTyrAla    180185190    GluGlnAspSerAsnHisCysPheIleSerThrValGluCysLysAla    195200205    ValMetValPhePheHisTyrCysValValSerAsnTyrPheTrpLeu    210215220    PheIleGluGlyLeuTyrLeuPheThrLeuLeuValGluThrPhePhe    225230235240    ProGluArgArgTyrPheTyrTrpTyrThrIleIleGlyTrpGlyThr    245250255    ProThrValCysValThrValTrpAlaThrLeuArgLeuTyrPheAsp    260265270    AspThrGlyCysTrpAspMetAsnAspSerThrAlaLeuTrpTrpVal    275280285    IleLysGlyProValValGlySerIleMetValAsnPheValLeuPhe    290295300    IleGlyIleIleValIleLeuValGlnLysLeuGlnSerProAspMet    305310315320    GlyGlyAsnGluSerSerIleTyrPheCysValGlnLysCysTyrCys    325330335    LysProGlnArgAlaGlnGlnHisSerCysLysMetSerGluLeuSer    340345350    ThrIleThrLeuArgLeuAlaArgSerThrLeuLeuLeuIleProLeu    355360365    PheGlyIleHisTyrThrValPheAlaPheSerProGluAsnValSer    370375380    LysArgGluArgLeuValPheGluLeuGlyLeuGlySerPheGlnGly    385390395400    PheValValAlaValLeuTyrCysPheLeuAsnGlyGluValGlnAla    405410415    GluIleLysArgLysTrpArgSerTrpLysValAsnArgTyrPheAla    420425430    ValAspPheLysHisArgHisProSerLeuAlaSerSerGlyValAsn    435440445    GlyGlyThrGlnLeuSerIleLeuSerLysSerSerSerGlnIleArg    450455460    MetSerGlyLeuProAlaAspAsnLeuAlaThr    465470475    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 552 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    MetAlaGlyValValHisValSerLeuAlaAlaHisCysGlyAlaCys    151015    ProTrpGlyArgGlyArgLeuArgLysGlyArgAlaAlaCysLysSer    202530    AlaAlaGlnArgHisIleGlyAlaAspLeuProLeuLeuSerValGly    354045    GlyGlnTrpCysTrpProArgSerValMetAlaGlyValValHisVal    505560    SerLeuAlaAlaLeuLeuLeuLeuProMetAlaProAlaMetHisSer    65707580    AspCysIlePheLysLysGluGlnAlaMetCysLeuGluLysIleGln    859095    ArgAlaAsnGluLeuMetGlyPheAsnAspSerSerProGlyCysPro    100105110    GlyMetTrpAspAsnIleThrCysTrpLysProAlaHisValGlyGlu    115120125    MetValLeuValSerCysProGluLeuPheArgIlePheAsnProAsp    130135140    GlnValTrpGluThrGluThrIleGlyGluSerAspPheGlyAspSer    145150155160    AsnSerLeuAspLeuSerAspMetGlyValValSerArgAsnCysThr    165170175    GluAspGlyTrpSerGluProPheProHisTyrPheAspAlaCysGly    180185190    PheAspGluTyrGluSerGluThrGlyAspGlnAspTyrTyrTyrLeu    195200205    SerValLysAlaLeuTyrThrValGlyTyrSerThrSerLeuValThr    210215220    LeuThrThrAlaMetValIleLeuCysArgPheArgLysLeuHisCys    225230235240    ThrArgAsnPheIleHisMetAsnLeuPheValSerPheMetLeuArg    245250255    AlaIleSerValPheIleLysAspTrpIleLeuTyrAlaGluGlnAsp    260265270    SerAsnHisCysPheIleSerThrValGluCysLysAlaValMetVal    275280285    PhePheHisTyrCysValValSerAsnTyrPheTrpLeuPheIleGlu    290295300    GlyLeuTyrLeuPheThrLeuLeuValGluThrPhePheProGluArg    305310315320    ArgTyrPheTyrTrpTyrThrIleIleGlyTrpGlyThrProThrVal    325330335    CysValThrValTrpAlaThrLeuArgLeuTyrPheAspAspThrGly    340345350    CysTrpAspMetAsnAspSerThrAlaLeuTrpTrpValIleLysGly    355360365    ProValValGlySerIleMetValAsnPheValLeuPheIleGlyIle    370375380    IleValIleLeuValGlnLysLeuGlnSerProAspMetGlyGlyAsn    385390395400    GluSerSerIleTyrPheCysValGlnLysCysTyrCysLysProGln    405410415    ArgAlaGlnGlnHisSerCysLysMetSerGluLeuSerThrIleThr    420425430    LeuArgLeuAlaArgSerThrLeuLeuLeuIleProLeuPheGlyIle    435440445    HisTyrThrValPheAlaPheSerProGluAsnValSerLysArgGlu    450455460    ArgLeuValPheGluLeuGlyLeuGlySerPheGlnGlyPheValVal    465470475480    AlaValLeuTyrCysPheLeuAsnGlyGluValGlnAlaGluIleLys    485490495    ArgLysTrpArgSerTrpLysValAsnArgTyrPheAlaValAspPhe    500505510    LysHisArgHisProSerLeuAlaSerSerGlyValAsnGlyGlyThr    515520525    GlnLeuSerIleLeuSerLysSerSerSerGlnIleArgMetSerGly    530535540    LeuProAlaAspAsnLeuAlaThr    545550    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 476 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    MetHisSerAspCysIlePheLysLysGluGlnAlaMetCysLeuGlu    151015    LysIleGlnArgAlaAsnGluLeuMetGlyPheAsnAspSerSerPro    202530    GlyCysProGlyMetTrpAspAsnIleThrCysTrpLysProAlaHis    354045    ValGlyGluMetValLeuValSerCysProGluLeuPheArgIlePhe    505560    AsnProAspGlnValTrpGluThrGluThrIleGlyGluSerAspPhe    65707580    GlyAspSerAsnSerLeuAspLeuSerAspMetGlyValValSerArg    859095    AsnCysThrGluAspGlyTrpSerGluProPheProHisTyrPheAsp    100105110    AlaCysGlyPheAspGluTyrGluSerGluThrGlyAspGlnAspTyr    115120125    TyrTyrLeuSerValLysAlaLeuTyrThrValGlyTyrSerThrSer    130135140    LeuValThrLeuThrThrAlaMetValIleLeuCysArgPheArgLys    145150155160    LeuHisCysThrArgAsnPheIleHisMetAsnLeuPheValSerPhe    165170175    MetLeuArgAlaIleSerValPheIleLysAspTrpIleLeuTyrAla    180185190    GluGlnAspSerAsnHisCysPheIleSerThrValGluCysLysAla    195200205    ValMetValPhePheHisTyrCysValValSerAsnTyrPheTrpLeu    210215220    PheIleGluGlyLeuTyrLeuPheThrLeuLeuValGluThrPhePhe    225230235240    ProGluArgArgTyrPheTyrTrpTyrThrIleIleGlyTrpGlyThr    245250255    ProThrValCysValThrValTrpAlaThrLeuArgLeuTyrPheAsp    260265270    AspThrGlyCysTrpAspMetAsnAspSerThrAlaLeuTrpTrpVal    275280285    IleLysGlyProValValGlySerIleMetValAsnPheValLeuPhe    290295300    IleGlyIleIleValIleLeuValGlnLysLeuGlnSerProAspMet    305310315320    GlyGlyAsnGluSerSerIleTyrLeuThrAsnLeuSerProArgVal    325330335    ProLysLysAlaArgGluAspProLeuProValProSerAspGlnHis    340345350    SerLeuProPheLeuArgLeuAlaArgSerThrLeuLeuLeuIlePro    355360365    LeuPheGlyIleHisTyrThrValPheAlaPheSerProGluAsnVal    370375380    SerLysArgGluArgLeuValPheGluLeuGlyLeuGlySerPheGln    385390395400    GlyPheValValAlaValLeuTyrCysPheLeuAsnGlyGluValGln    405410415    AlaGluIleLysArgLysTrpArgSerTrpLysValAsnArgTyrPhe    420425430    AlaValAspPheLysHisArgHisProSerLeuAlaSerSerGlyVal    435440445    AsnGlyGlyThrGlnLeuSerIleLeuSerLysSerSerSerGlnIle    450455460    ArgMetSerGlyLeuProAlaAspAsnLeuAlaThr    465470475    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 553 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    MetAlaGlyValValHisValSerLeuAlaAlaHisCysGlyAlaCys    151015    ProTrpGlyArgGlyArgLeuArgLysGlyArgAlaAlaCysLysSer    202530    AlaAlaGlnArgHisIleGlyAlaAspLeuProLeuLeuSerValGly    354045    GlyGlnTrpCysTrpProArgSerValMetAlaGlyValValHisVal    505560    SerLeuAlaAlaLeuLeuLeuLeuProMetAlaProAlaMetHisSer    65707580    AspCysIlePheLysLysGluGlnAlaMetCysLeuGluLysIleGln    859095    ArgAlaAsnGluLeuMetGlyPheAsnAspSerSerProGlyCysPro    100105110    GlyMetTrpAspAsnIleThrCysTrpLysProAlaHisValGlyGlu    115120125    MetValLeuValSerCysProGluLeuPheArgIlePheAsnProAsp    130135140    GlnValTrpGluThrGluThrIleGlyGluSerAspPheGlyAspSer    145150155160    AsnSerLeuAspLeuSerAspMetGlyValValSerArgAsnCysThr    165170175    GluAspGlyTrpSerGluProPheProHisTyrPheAspAlaCysGly    180185190    PheAspGluTyrGluSerGluThrGlyAspGlnAspTyrTyrTyrLeu    195200205    SerValLysAlaLeuTyrThrValGlyTyrSerThrSerLeuValThr    210215220    LeuThrThrAlaMetValIleLeuCysArgPheArgLysLeuHisCys    225230235240    ThrArgAsnPheIleHisMetAsnLeuPheValSerPheMetLeuArg    245250255    AlaIleSerValPheIleLysAspTrpIleLeuTyrAlaGluGlnAsp    260265270    SerAsnHisCysPheIleSerThrValGluCysLysAlaValMetVal    275280285    PhePheHisTyrCysValValSerAsnTyrPheTrpLeuPheIleGlu    290295300    GlyLeuTyrLeuPheThrLeuLeuValGluThrPhePheProGluArg    305310315320    ArgTyrPheTyrTrpTyrThrIleIleGlyTrpGlyThrProThrVal    325330335    CysValThrValTrpAlaThrLeuArgLeuTyrPheAspAspThrGly    340345350    CysTrpAspMetAsnAspSerThrAlaLeuTrpTrpValIleLysGly    355360365    ProValValGlySerIleMetValAsnPheValLeuPheIleGlyIle    370375380    IleValIleLeuValGlnLysLeuGlnSerProAspMetGlyGlyAsn    385390395400    GluSerSerIleTyrLeuThrAsnLeuSerProArgValProLysLys    405410415    AlaArgGluAspProLeuProValProSerAspGlnHisSerLeuPro    420425430    PheLeuArgLeuAlaArgSerThrLeuLeuLeuIleProLeuPheGly    435440445    IleHisTyrThrValPheAlaPheSerProGluAsnValSerLysArg    450455460    GluArgLeuValPheGluLeuGlyLeuGlySerPheGlnGlyPheVal    465470475480    ValAlaValLeuTyrCysPheLeuAsnGlyGluValGlnAlaGluIle    485490495    LysArgLysTrpArgSerTrpLysValAsnArgTyrPheAlaValAsp    500505510    PheLysHisArgHisProSerLeuAlaSerSerGlyValAsnGlyGly    515520525    ThrGlnLeuSerIleLeuSerLysSerSerSerGlnIleArgMetSer    530535540    GlyLeuProAlaAspAsnLeuAlaThr    545550    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1539 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 1..1539    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    ATGAGAGGCGGGCGGCACTGGCCCGAGCCGCCTTGCAGGCTGAGAAGCGTCATGGCCAGC60    ATCGCGCAGGTCTCCCTGGCTGCTCTCCTCCTGCTGCCTATGGCCACCGCCATGCATTCC120    GACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGATCCAGAGGGTGAATGAC180    CTGATGGGCTTGAATGACTCCTCCCCAGGGTGCCCTGGGATGTGGGACAACATCACGTGT240    TGGAAGCCCGCCCACGTGGGTGAGATGGTCCTGGTCAGTTGCCCTGAACTCTTCCGAATC300    TTCAACCCAGACCAAGTCTGGGAGACGGAAACCATCGGAGAGTTCGGTTTTGCAGACAGT360    AAATCCTTGGATCTCTCAGACATGAGGGTGGTGAGCCGGAATTGCACGGAGGATGGATGG420    TCAGAGCCATTCCCTCATTATTTCGATGCCTGTGGGTTTGAGGAGTACGAATCTGAGACT480    GGGGACCAGGATTACTACTACCTGTCAGTGAAGGCCCTGTACACAGTTGGCTACAGCACG540    TCCCTCGTCACCCTCACCACTGCCATGGTCATCCTGTGTCGTTTCCGGAAGCTGCACTGC600    ACCCGCAACTTCATCCACATGAACCTCTTCGTGTCGTTTATGCTGAGGGCCATCTCCGTC660    TTCATCAAAGACTGGATCCTCTATGCTGAGCAGGACAGCAATCACTGCTTTGTCTCCACT720    GTGGAATGCAAGGCTGTGATGGTTTTCTTCCACTACTGTGTTGTATCCAACTACTTCTGG780    CTGTTCATCGAGGGCCTGTATCTCTTCACCCTGCTGGTGGAGACCTTCTTCCCCGAGAGG840    AGATATTTCTACTGGTACATCATCATTGGCTGGGGGACACCAACTGTGTGTGTGTCTGTG900    TGGGCTATGCTGAGGCTCTACTTCGATGACACAGGCTGCTGGGATATGAATGACAACACG960    GCTCTGTGGTGGGTGATCAAAGGCCCTGTAGTTGGCTCCATAATGGTTAATTTTGTGCTC1020    TTCATCGGCATCATTGTCATCCTTGTGCAGAAACTTCAGTCTCCAGACATGGGAGGCAAC1080    GAGTCCAGCATCTACTTCAGCTGCGTGCAGAAATGCTACTGCAAGCCACAGCGGGCTCAG1140    CAGCACTCTTGCAAGATGTCAGAACTGTCCACCATTACTCTACGGCTCGCCAGGTCCACC1200    TTGCTGCTCATCCCACTCTTTGGAATCCACTACACTGTCTTTGCTTTCTCCCCGGAGAAC1260    GTCAGCAAGAGGGAGAGACTGGTGTTTGAGCTGGGTCTGGGCTCCTTCCAGGGCTTTGTG1320    GTGGCTGTTCTCTATTGCTTTCTGAATGGAGAGGTGCAGGCGGAGATCAAGAGGAAGTGG1380    CGGAGCTGGAAGGTGAACCGCTACTTCACCATGGACTTCAAGCACCGGCACCCATCCCTG1440    GCCAGCAGCGGGGTGAACGGGGGCACCCAGCTCTCCATCCTGAGCAAGAGCAGCTCCCAG1500    ATCCGCATGTCTGGGCTTCCGGCCGACAACCTGGCCACC1539    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1455 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 1..1455    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    ATGAGAGGCGGGCGGCACTGGCCCGAGCCGCCTTGCAGGCTGAGAAGCGTCATGGCCAGC60    ATCGCGCAGGTCTCCCTGGCTGCTCTCCTCCTGCTGCCTATGGCCACCGCCATGCATTCC120    GACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGATCCAGAGGGTGAATGAC180    CTGATGGGCTTGAATGACTCCTCCCCAGGGTGCCCTGGGATGTGGGACAACATCACGTGT240    TGGAAGCCCGCCCACGTGGGTGAGATGGTCCTGGTCAGTTGCCCTGAACTCTTCCGAATC300    TTCAACCCAGACCAAGTCTGGGAGACGGAAACCATCGGAGAGTTCGGTTTTGCAGACAGT360    AAATCCTTGGATCTCTCAGACATGAGGGTGGTGAGCCGGAATTGCACGGAGGATGGATGG420    TCAGAGCCATTCCCTCATTATTTCGATGCCTGTGGGTTTGAGGAGTACGAATCTGAGACT480    GGGGACCAGGATTACTACTACCTGTCAGTGAAGGCCCTGTACACAGTTGGCTACAGCACG540    TCCCTCGTCACCCTCACCACTGCCATGGTCATCCTGTGTCGTTTCCGGAAGCTGCACTGC600    ACCCGCAACTTCATCCACATGAACCTCTTCGTGTCGTTTATGCTGAGGGCCATCTCCGTC660    TTCATCAAAGACTGGATCCTCTATGCTGAGCAGGACAGCAATCACTGCTTTGTCTCCACT720    GTGGAATGCAAGGCTGTGATGGTTTTCTTCCACTACTGTGTTGTATCCAACTACTTCTGG780    CTGTTCATCGAGGGCCTGTATCTCTTCACCCTGCTGGTGGAGACCTTCTTCCCCGAGAGG840    AGATATTTCTACTGGTACATCATCATTGGCTGGGGGACACCAACTGTGTGTGTGTCTGTG900    TGGGCTATGCTGAGGCTCTACTTCGATGACACAGGCTGCTGGGATATGAATGACAACACG960    GCTCTGTGGTGGGTGATCAAAGGCCCTGTAGTTGGCTCCATAATGGTTAATTTTGTGCTC1020    TTCATCGGCATCATTGTCATCCTTGTGCAGAAACTTCAGTCTCCAGACATGGGAGGCAAC1080    GAGTCCAGCATCTACTTACGGCTCGCCAGGTCCACCTTGCTGCTCATCCCACTCTTTGGA1140    ATCCACTACACTGTCTTTGCTTTCTCCCCGGAGAACGTCAGCAAGAGGGAGAGACTGGTG1200    TTTGAGCTGGGTCTGGGCTCCTTCCAGGGCTTTGTGGTGGCTGTTCTCTATTGCTTTCTG1260    AATGGAGAGGTGCAGGCGGAGATCAAGAGGAAGTGGCGGAGCTGGAAGGTGAACCGCTAC1320    TTCACCATGGACTTCAAGCACCGGCACCCATCCCTGGCCAGCAGCGGGGTGAACGGGGGC1380    ACCCAGCTCTCCATCCTGAGCAAGAGCAGCTCCCAGATCCGCATGTCTGGGCTTCCGGCC1440    GACAACCTGGCCACC1455    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1401 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 1..1401    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    ATGGCCAGAGTCCTGCAGCTCTCCCTGACTGCTCTCCTGCTGCCTGTGGCTATTGCTATG60    CACTCTGACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAGGATCCAGAGGGCC120    AACGACCTGATGGGACTAAACGAGTCTTCCCCAGGTTGCCCTGGCATGTGGGACAATATC180    ACATGTTGGAAGCCAGCTCAAGTAGGTGAGATGGTCCTTGTAAGCTGCCCTGAGGTCTTC240    CGGATCTTCAACCCGGACCAAGTCTGGATGACAGAAACCATAGGAGATTCTGGTTTTGCC300    GATAGTAATTCCTTGGAGATCACAGACATGGGGGTCGTGGGCCGGAACTGCACAGAGGAC360    GGCTGGTCGGAGCCCTTCCCCCACTACTTCGATGCTTGTGGGTTTGATGATTATGAGCCT420    GAGTCTGGAGATCAGGATTATTACTACCTGTCGGTGAAGGCTCTCTACACAGTCGGCTAC480    AGCACTTCCCTCGCCACCCTCACTACTGCCATGGTCATCTTGTGCCGCTTCCGGAAGCTG540    CATTGCACTCGCAACTTCATCCACATGAACCTGTTTGTATCCTTCATGCTGAGGGCTATC600    TCCGTCTTCATCAAGGACTGGATCTTGTACGCCGAGCAGGACAGCAGTCACTGCTTCGTT660    TCCACCGTGGAGTGCAAAGCTGTCATGGTTTTCTTCCACTACTGCGTGGTGTCCAACTAC720    TTCTGGCTGTTCATTGAAGGCCTGTACCTCTTTACACTGCTGGTGGAGACCTTCTTCCCT780    GAGAGGAGATATTTCTACTGGTACACCATCATCGGCTGGGGGACACCTACTGTGTGTGTA840    ACAGTGTGGGCTGTGCTGAGGCTCTATTTTGATGATGCAGGATGCTGGGATATGAATGAC900    AGCACAGCTCTGTGGTGGGTGATCAAAGGCCCCGTGGTTGGCTCTATAATGGTTAACTTT960    GTGCTTTTCATCGGCATCATCATCATCCTTGTACAGAAGCTGCAGTCCCCAGACATGGGA1020    GGCAACGAGTCCAGCATCTACTTACGGCTGGCCCGCTCCACCCTACTGCTCATCCCACTC1080    TTCGGAATCCACTACACAGTATTCGCCTTCTCTCCAGAGAACGTCAGCAAGAGGGAAAGA1140    CTTGTGTTTGAGCTTGGGCTGGGCTCCTTCCAGGGCTTTGTGGTGGCTGTACTCTACTGC1200    TTCCTGAATGGGGAGGTACAGGCAGAGATTAAGAGGAAATGGAGGAGCTGGAAGGTGAAC1260    CGTTACTTCACTATGGACTTCAAGCACCGGCACCCGTCCCTGGCCAGCAGTGGAGTAAAT1320    GGGGGAACCCAGCTGTCCATCCTGAGCAAGAGCAGCTCCCAGCTCCGCATGTCCAGCCTC1380    CCGGCCGACAACTTGGCCACC1401    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1485 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 1..1485    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    ATGGCCAGAGTCCTGCAGCTCTCCCTGACTGCTCTCCTGCTGCCTGTGGCTATTGCTATG60    CACTCTGACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAGGATCCAGAGGGCC120    AACGACCTGATGGGACTAAACGAGTCTTCCCCAGGTTGCCCTGGCATGTGGGACAATATC180    ACATGTTGGAAGCCAGCTCAAGTAGGTGAGATGGTCCTTGTAAGCTGCCCTGAGGTCTTC240    CGGATCTTCAACCCGGACCAAGTCTGGATGACAGAAACCATAGGAGATTCTGGTTTTGCC300    GATAGTAATTCCTTGGAGATCACAGACATGGGGGTCGTGGGCCGGAACTGCACAGAGGAC360    GGCTGGTCGGAGCCCTTCCCCCACTACTTCGATGCTTGTGGGTTTGATGATTATGAGCCT420    GAGTCTGGAGATCAGGATTATTACTACCTGTCGGTGAAGGCTCTCTACACAGTCGGCTAC480    AGCACTTCCCTCGCCACCCTCACTACTGCCATGGTCATCTTGTGCCGCTTCCGGAAGCTG540    CATTGCACTCGCAACTTCATCCACATGAACCTGTTTGTATCCTTCATGCTGAGGGCTATC600    TCCGTCTTCATCAAGGACTGGATCTTGTACGCCGAGCAGGACAGCAGTCACTGCTTCGTT660    TCCACCGTGGAGTGCAAAGCTGTCATGGTTTTCTTCCACTACTGCGTGGTGTCCAACTAC720    TTCTGGCTGTTCATTGAAGGCCTGTACCTCTTTACACTGCTGGTGGAGACCTTCTTCCCT780    GAGAGGAGATATTTCTACTGGTACACCATCATCGGCTGGGGGACACCTACTGTGTGTGTA840    ACAGTGTGGGCTGTGCTGAGGCTCTATTTTGATGATGCAGGATGCTGGGATATGAATGAC900    AGCACAGCTCTGTGGTGGGTGATCAAAGGCCCCGTGGTTGGCTCTATAATGGTTAACTTT960    GTGCTTTTCATCGGCATCATCATCATCCTTGTACAGAAGCTGCAGTCCCCAGACATGGGA1020    GGCAACGAGTCCAGCATCTACTTCAGCTGCGTGCAGAAATGCTACTGCAAGCCACAGCGG1080    GCTCAGCAGCACTCTTGCAAGATGTCAGAACTATCCACCATTACTCTACGGCTGGCCCGC1140    TCCACCCTACTGCTCATCCCACTCTTCGGAATCCACTACACAGTATTCGCCTTCTCTCCA1200    GAGAACGTCAGCAAGAGGGAAAGACTTGTGTTTGAGCTTGGGCTGGGCTCCTTCCAGGGC1260    TTTGTGGTGGCTGTACTCTACTGCTTCCTGAATGGGGAGGTACAGGCAGAGATTAAGAGG1320    AAATGGAGGAGCTGGAAGGTGAACCGTTACTTCACTATGGACTTCAAGCACCGGCACCCG1380    TCCCTGGCCAGCAGTGGAGTAAATGGGGGAACCCAGCTGTCCATCCTGAGCAAGAGCAGC1440    TCCCAGCTCCGCATGTCCAGCCTCCCGGCCGACAACTTGGCCACC1485    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1575 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 1..1575    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    ATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCACTGCGGGGCCTGTCCGTGGGGCCGG60    GGCAGACTCCGCAAAGGACGCGCAGCCTGCAAGTCCGCGGCCCAGAGACACATTGGGGCT120    GACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTGGCCAAGAAGTGTCATGGCTGGT180    GTCGTGCACGTTTCCCTGGCTGCTCTCCTCCTGCTGCCTATGGCCCCTGCCATGCATTCT240    GACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGATCCAGAGGGCCAATGAG300    CTGATGGGCTTCAATGATTCCTCTCCAGGCTGTCCTGGGATGTGGGACAACATCACGTGT360    TGGAAGCCCGCCCATGTGGGTGAGATGGTCCTGGTCAGCTGCCCTGAGCTCTTCCGAATC420    TTCAACCCAGACCAAGTCTGGGAGACCGAAACCATTGGAGAGTCTGATTTTGGTGACAGT480    AACTCCTTAGATCTCTCAGACATGGGAGTGGTGAGCCGGAACTGCACGGAGGATGGCTGG540    TCGGAACCCTTCCCTCATTACTTTGATGCCTGTGGGTTTGATGAATATGAATCTGAGACT600    GGGGACCAGGATTATTACTACCTGTCAGTGAAGGCCCTCTACACGGTTGGCTACAGCACA660    TCCCTCGTCACCCTCACCACTGCCATGGTCATCCTTTGTCGCTTCCGGAAGCTGCACTGC720    ACACGCAACTTCATCCACATGAACCTGTTTGTGTCGTTCATGCTGAGGGCGATCTCCGTC780    TTCATCAAAGACTGGATTCTGTATGCGGAGCAGGACAGCAACCACTGCTTCATCTCCACT840    GTGGAATGTAAGGCCGTCATGGTTTTCTTCCACTACTGTGTTGTGTCCAACTACTTCTGG900    CTGTTCATCGAGGGCCTGTACCTCTTCACTCTGCTGGTGGAGACCTTCTTCCCTGAAAGG960    AGATACTTCTACTGGTACACCATCATTGGCTGGGGGTCCCCAACTGTGTGTGTGACAGTG1020    TGGGCTACGCTGAGACTCTACTTTGATGACACAGGCTGCTGGGATATGAATGACAGCACA1080    GCTCTGTGGTGGGTGATCAAAGGCCCTGTGGTTGGCTCTATCATGGTTAACTTTGTGCTT1140    TTTATTGGCATTATCGTCATCCTTGTGCAGAAACTTCAGTCTCCAGACATGGGAGGCAAT1200    GAGTCCAGCATCTACTTGCGACTGGCCCGGTCCACCCTGCTGCTCATCCCACTATTCGGA1260    ATCCACTACACAGTATTTGCCTTCTCCCCAGAGAATGTCAGCAAAAGGGAAAGACTCGTG1320    TTTGAGCTGGGGCTGGGCTCCTTCCAGGGCTTTGTGGTGGCTGTTCTCTACTGTTTTCTG1380    AATGGTGAGGTACAAGCGGAGATCAAGCGAAAATGGCGAAGCTGGAAGGTGAACCGTTAC1440    TTCGCTGTGGACTTCAAGCACCGACACCCGTCTCTGGCCAGCAGTGGGGTGAATGGGGGC1500    ACCCAGCTCTCCATCCTGAGCAAGAGCAGCTCCCAAATCCGCATGTCTGGCCTCCCTGCT1560    GACAATCTGGCCACC1575    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1659 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 1..1659    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    ATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCACTGCGGGGCCTGTCCGTGGGGCCGG60    GGCAGACTCCGCAAAGGACGCGCAGCCTGCAAGTCCGCGGCCCAGAGACACATTGGGGCT120    GACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTGGCCAAGAAGTGTCATGGCTGGT180    GTCGTGCACGTTTCCCTGGCTGCTCTCCTCCTGCTGCCTATGGCCCCTGCCATGCATTCT240    GACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGATCCAGAGGGCCAATGAG300    CTGATGGGCTTCAATGATTCCTCTCCAGGCTGTCCTGGGATGTGGGACAACATCACGTGT360    TGGAAGCCCGCCCATGTGGGTGAGATGGTCCTGGTCAGCTGCCCTGAGCTCTTCCGAATC420    TTCAACCCAGACCAAGTCTGGGAGACCGAAACCATTGGAGAGTCTGATTTTGGTGACAGT480    AACTCCTTAGATCTCTCAGACATGGGAGTGGTGAGCCGGAACTGCACGGAGGATGGCTGG540    TCGGAACCCTTCCCTCATTACTTTGATGCCTGTGGGTTTGATGAATATGAATCTGAGACT600    GGGGACCAGGATTATTACTACCTGTCAGTGAAGGCCCTCTACACGGTTGGCTACAGCACA660    TCCCTCGTCACCCTCACCACTGCCATGGTCATCCTTTGTCGCTTCCGGAAGCTGCACTGC720    ACACGCAACTTCATCCACATGAACCTGTTTGTGTCGTTCATGCTGAGGGCGATCTCCGTC780    TTCATCAAAGACTGGATTCTGTATGCGGAGCAGGACAGCAACCACTGCTTCATCTCCACT840    GTGGAATGTAAGGCCGTCATGGTTTTCTTCCACTACTGTGTTGTGTCCAACTACTTCTGG900    CTGTTCATCGAGGGCCTGTACCTCTTCACTCTGCTGGTGGAGACCTTCTTCCCTGAAAGG960    AGATACTTCTACTGGTACACCATCATTGGCTGGGGGACCCCAACTGTGTGTGTGACAGTG1020    TGGGCTACGCTGAGACTCTACTTTGATGACACAGGCTGCTGGGATATGAATGACAGCACA1080    GCTCTGTGGTGGGTGATCAAAGGCCCTGTGGTTGGCTCTATCATGGTTAACTTTGTGCTT1140    TTTATTGGCATTATCGTCATCCTTGTGCAGAAACTTCAGTCTCCAGACATGGGAGGCAAT1200    GAGTCCAGCATCTACTTCAGCTGCGTGCAGAAATGCTACTGCAAGCCACAGCGGGCTCAG1260    CAGCACTCTTGCAAGATGTCAGAACTGTCCACCATTACTCTGCGACTGGCCCGGTCCACC1320    CTGCTGCTCATCCCACTATTCGGAATCCACTACACAGTATTTGCCTTCTCCCCAGAGAAT1380    GTCAGCAAAAGGGAAAGACTCGTGTTTGAGCTGGGGCTGGGCTCCTTCCAGGGCTTTGTG1440    GTGGCTGTTCTCTACTGTTTTCTGAATGGTGAGGTACAAGCGGAGATCAAGCGAAAATGG1500    CGAAGCTGGAAGGTGAACCGTTACTTCGCTGTGGACTTCAAGCACCGACACCCGTCTCTG1560    GCCAGCAGTGGGGTGAATGGGGGCACCCAGCTCTCCATCCTGAGCAAGAGCAGCTCCCAA1620    ATCCGCATGTCTGGCCTCCCTGCTGACAATCTGGCCACC1659    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1656 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 1..1656    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    ATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCACTGCGGGGCCTGTCCGTGGGGCCGG60    GGCAGACTCCGCAAAGGACGCGCAGCCTGCAAGTCCGCGGCCCAGAGACACATTGGGGCT120    GACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTGGCCAAGAAGTGTCATGGCTGGT180    GTCGTGCACGTTTCCCTGGCTGCTCTCCTCCTGCTGCCTATGGCCCCTGCCATGCATTCT240    GACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGATCCAGAGGGCCAATGAG300    CTGATGGGCTTCAATGATTCCTCTCCAGGCTGTCCTGGGATGTGGGACAACATCACGTGT360    TGGAAGCCCGCCCATGTGGGTGAGATGGTCCTGGTCAGCTGCCCTGAGCTCTTCCGAATC420    TTCAACCCAGACCAAGTCTGGGAGACCGAAACCATTGGAGAGTCTGATTTTGGTGACAGT480    AACTCCTTAGATCTCTCAGACATGGGAGTGGTGAGCCGGAACTGCACGGAGGATGGCTGG540    TCGGAACCCTTCCCTCATTACTTTGATGCCTGTGGGTTTGATGAATATGAATCTGAGACT600    GGGGACCAGGATTATTACTACCTGTCAGTGAAGGCCCTCTACACGGTTGGCTACAGCACA660    TCCCTCGTCACCCTCACCACTGCCATGGTCATCCTTTGTCGCTTCCGGAAGCTGCACTGC720    ACACGCAACTTCATCCACATGAACCTGTTTGTGTCGTTCATGCTGAGGGCGATCTCCGTC780    TTCATCAAAGACTGGATTCTGTATGCGGAGCAGGACAGCAACCACTGCTTCATCTCCACT840    GTGGAATGTAAGGCCGTCATGGTTTTCTTCCACTACTGTGTTGTGTCCAACTACTTCTGG900    CTGTTCATCGAGGGCCTGTACCTCTTCACTCTGCTGGTGGAGACCTTCTTCCCTGAAAGG960    AGATACTTCTACTGGTACACCATCATTGGCTGGGGGACCCCAACTGTGTGTGTGACAGTG1020    TGGGCTACGCTGAGACTCTACTTTGATGACACAGGCTGCTGGGATATGAATGACAGCACA1080    GCTCTGTGGTGGGTGATCAAAGGCCCTGTGGTTGGCTCTATCATGGTTAACTTTGTGCTT1140    TTTATTGGCATTATCGTCATCCTTGTGCAGAAACTTCAGTCTCCAGACATGGGAGGCAAT1200    GAGTCCAGCATCTACTTCTGCGTGCAGAAATGCTACTGCAAGCCACAGCGGGCTCAGCAG1260    CACTCTTGCAAGATGTCAGAACTGTCCACCATTACTCTGCGACTGGCCCGGTCCACCCTG1320    CTGCTCATCCCACTATTCGGAATCCACTACACAGTATTTGCCTTCTCCCCAGAGAATGTC1380    AGCAAAAGGGAAAGACTCGTGTTTGAGCTGGGGCTGGGCTCCTTCCAGGGCTTTGTGGTG1440    GCTGTTCTCTACTGTTTTCTGAATGGTGAGGTACAAGCGGAGATCAAGCGAAAATGGCGA1500    AGCTGGAAGGTGAACCGTTACTTCGCTGTGGACTTCAAGCACCGACACCCGTCTCTGGCC1560    AGCAGTGGGGTGAATGGGGGCACCCAGCTCTCCATCCTGAGCAAGAGCAGCTCCCAAATC1620    CGCATGTCTGGCCTCCCTGCTGACAATCTGGCCACC1656    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1659 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 1..1659    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    ATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCACTGCGGGGCCTGTCCGTGGGGCCGG60    GGCAGACTCCGCAAAGGACGCGCAGCCTGCAAGTCCGCGGCCCAGAGACACATTGGGGCT120    GACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTGGCCAAGAAGTGTCATGGCTGGT180    GTCGTGCACGTTTCCCTGGCTGCTCTCCTCCTGCTGCCTATGGCCCCTGCCATGCATTCT240    GACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGATCCAGAGGGCCAATGAG300    CTGATGGGCTTCAATGATTCCTCTCCAGGCTGTCCTGGGATGTGGGACAACATCACGTGT360    TGGAAGCCCGCCCATGTGGGTGAGATGGTCCTGGTCAGCTGCCCTGAGCTCTTCCGAATC420    TTCAACCCAGACCAAGTCTGGGAGACCGAAACCATTGGAGAGTCTGATTTTGGTGACAGT480    AACTCCTTAGATCTCTCAGACATGGGAGTGGTGAGCCGGAACTGCACGGAGGATGGCTGG540    TCGGAACCCTTCCCTCATTACTTTGATGCCTGTGGGTTTGATGAATATGAATCTGAGACT600    GGGGACCAGGATTATTACTACCTGTCAGTGAAGGCCCTCTACACGGTTGGCTACAGCACA660    TCCCTCGTCACCCTCACCACTGCCATGGTCATCCTTTGTCGCTTCCGGAAGCTGCACTGC720    ACACGCAACTTCATCCACATGAACCTGTTTGTGTCGTTCATGCTGAGGGCGATCTCCGTC780    TTCATCAAAGACTGGATTCTGTATGCGGAGCAGGACAGCAACCACTGCTTCATCTCCACT840    GTGGAATGTAAGGCCGTCATGGTTTTCTTCCACTACTGTGTTGTGTCCAACTACTTCTGG900    CTGTTCATCGAGGGCCTGTACCTCTTCACTCTGCTGGTGGAGACCTTCTTCCCTGAAAGG960    AGATACTTCTACTGGTACACCATCATTGGCTGGGGGACCCCAACTGTGTGTGTGACAGTG1020    TGGGCTACGCTGAGACTCTACTTTGATGACACAGGCTGCTGGGATATGAATGACAGCACA1080    GCTCTGTGGTGGGTGATCAAAGGCCCTGTGGTTGGCTCTATCATGGTTAACTTTGTGCTT1140    TTTATTGGCATTATCGTCATCCTTGTGCAGAAACTTCAGTCTCCAGACATGGGAGGCAAT1200    GAGTCCAGCATCTACTTAACAAATTTAAGCCCGCGAGTCCCCAAGAAAGCCCGAGAGGAC1260    CCCCTGCCTGTGCCCTCAGACCAGCATTCACTCCCTTTCCTGCGACTGGCCCGGTCCACC1320    CTGCTGCTCATCCCACTATTCGGAATCCACTACACAGTATTTGCCTTCTCCCCAGAGAAT1380    GTCAGCAAAAGGGAAAGACTCGTGTTTGAGCTGGGGCTGGGCTCCTTCCAGGGCTTTGTG1440    GTGGCTGTTCTCTACTGTTTTCTGAATGGTGAGGTACAAGCGGAGATCAAGCGAAAATGG1500    CGAAGCTGGAAGGTGAACCGTTACTTCGCTGTGGACTTCAAGCACCGACACCCGTCTCTG1560    GCCAGCAGTGGGGTGAATGGGGGCACCCAGCTCTCCATCCTGAGCAAGAGCAGCTCCCAA1620    ATCCGCATGTCTGGCCTCCCTGCTGACAATCTGGCCACC1659    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2814 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 498..2036    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    TGGCCTGCACCCCACCCCCCAGCCTGCGAAGACGGGGGGAGGCGGTGGTCGGTCGCCTCC60    CTCCTGCCCCCGGCCTGGCTTCGCGGTGGAGGCGGTGCCTCTCCGGCAAGGCAGACCAGG120    CTGGGCGGACGCGCGGCGCGGGGCGGGCTAGGGAAGGCCGGGGGCCTCGCGCTCGGGCCC180    CGGGCGGCGACTGACAGCGGCGGCGGCGGCGGCAGCGGCTCCAAGGCGAGCGTGGTCCCC240    GCGTGCGCACAAGCTCGCCGCCGCGCAGGGACCCACGGACACCGGCGCCGGGCGGACACA300    CAGACGCGGAGATCGGGCTCTACGCGCGCTACTCAGCGCACGAGCTCCCCATCCCTGGGC360    GGAGCGGGGCGCGGACTCGCCGCTGCGCGCCCTCCCCGCGGAGTCTGCCCCGGGCAGACC420    CGCAGCCCGCGGCCCCGCCGCGAGGCCCCTGGGTGAGCAGCCTGTAGACACCTGGGGTTG480    AGCAGTGGCGGCTGTGAATGAGAGGCGGGCGGCACTGGCCCGAGCCGCCTTGCAGGCTGA540    GAAGCGTCATGGCCAGCATCGCGCAGGTCTCCCTGGCTGCTCTCCTCCTGCTGCCTATGG600    CCACCGCCATGCATTCCGACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGA660    TCCAGAGGGTGAATGACCTGATGGGCTTGAATGACTCCTCCCCAGGGTGCCCTGGGATGT720    GGGACAACATCACGTGTTGGAAGCCCGCCCACGTGGGTGAGATGGTCCTGGTCAGTTGCC780    CTGAACTCTTCCGAATCTTCAACCCAGACCAAGTCTGGGAGACGGAAACCATCGGAGAGT840    TCGGTTTTGCAGACAGTAAATCCTTGGATCTCTCAGACATGAGGGTGGTGAGCCGGAATT900    GCACGGAGGATGGATGGTCAGAGCCATTCCCTCATTATTTCGATGCCTGTGGGTTTGAGG960    AGTACGAATCTGAGACTGGGGACCAGGATTACTACTACCTGTCAGTGAAGGCCCTGTACA1020    CAGTTGGCTACAGCACGTCCCTCGTCACCCTCACCACTGCCATGGTCATCCTGTGTCGTT1080    TCCGGAAGCTGCACTGCACCCGCAACTTCATCCACATGAACCTCTTCGTGTCGTTTATGC1140    TGAGGGCCATCTCCGTCTTCATCAAAGACTGGATCCTCTATGCTGAGCAGGACAGCAATC1200    ACTGCTTTGTCTCCACTGTGGAATGCAAGGCTGTGATGGTTTTCTTCCACTACTGTGTTG1260    TATCCAACTACTTCTGGCTGTTCATCGAGGGCCTGTATCTCTTCACCCTGCTGGTGGAGA1320    CCTTCTTCCCCGAGAGGAGATATTTCTACTGGTACATCATCATTGGCTGGGGGACACCAA1380    CTGTGTGTGTGTCTGTGTGGGCTATGCTGAGGCTCTACTTCGATGACACAGGCTGCTGGG1440    ATATGAATGACAACACGGCTCTGTGGTGGGTGATCAAAGGCCCTGTAGTTGGCTCCATAA1500    TGGTTAATTTTGTGCTCTTCATCGGCATCATTGTCATCCTTGTGCAGAAACTTCAGTCTC1560    CAGACATGGGAGGCAACGAGTCCAGCATCTACTTCAGCTGCGTGCAGAAATGCTACTGCA1620    AGCCACAGCGGGCTCAGCAGCACTCTTGCAAGATGTCAGAACTGTCCACCATTACTCTAC1680    GGCTCGCCAGGTCCACCTTGCTGCTCATCCCACTCTTTGGAATCCACTACACTGTCTTTG1740    CTTTCTCCCCGGAGAACGTCAGCAAGAGGGAGAGACTGGTGTTTGAGCTGGGTCTGGGCT1800    CCTTCCAGGGCTTTGTGGTGGCTGTTCTCTATTGCTTTCTGAATGGAGAGGTGCAGGCGG1860    AGATCAAGAGGAAGTGGCGGAGCTGGAAGGTGAACCGCTACTTCACCATGGACTTCAAGC1920    ACCGGCACCCATCCCTGGCCAGCAGCGGGGTGAACGGGGGCACCCAGCTCTCCATCCTGA1980    GCAAGAGCAGCTCCCAGATCCGCATGTCTGGGCTTCCGGCCGACAACCTGGCCACCTGAG2040    CCCACCCTGCCCCCTCCTCTCCTCTGTACGCAGGCTGGGGCTGTGGTGGGGCGCCGGCCC2100    ACGCATGTTGTGCCTCTTCTCGCCTTCGGGCAGGCCCCGGGCTGGGCGCCTGGCCCCCGA2160    GGTTGGAGAAGGATGCGGGACAGGCAGCTGTTTAGCCTTCCTGTTTTGGCGCTGGCCCAA2220    CCACCGTGGGTCCCTGGGCCTGCACCCAGACATGTAATACTCCTTAATTGGGAAGTCATC2280    CATTCTTTCCCTTTCCCAAGTCCTTGCTTATTAAGAGGTTCAAGTCACCTACCCAATTCA2340    GAAGCTTAAGTAACCACTAACCACCGTGACTGCGTGGGAGGCCTCCCATGGGCTGAGCTA2400    CTGACTTGGCTTTGGGGGCCTTGGGCTGGGGCCCTCCTTAAAGCCCCCCCTGAAATTGTC2460    GGACCTCAAAGTGTGACTCCTTTGAGTCTACTCGCCACCCCCGTGGCCCTTTGCAGCCCT2520    GGTCCAGTCACCGAGGTTACTGGAAGTCCAGCTTGGATGGCCAGACAGCTTTTTGGCACA2580    GGCAGACCCATGCTCACCCAACATTTTAGTGTCCAGGTGCCCAGGTGCCCAGGTGCCCAG2640    CTCCTGGGCATCAGACAGTGGGAAAGCTCCAGGGATCTACCATTCAGAGACTTCAGTTTG2700    GATGTAGGGCTAAGGCCAGAGAAAAGTTCTGGAGCTTTTCATTTGGCCCAAGAAAAAACT2760    GCCAAGATCCAGAAAAGTGGATCTGAGTGGAATTTAGATGCAAAGAGCTTGGAG2814    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2730 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 498..1952    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    TGGCCTGCACCCCACCCCCCAGCCTGCGAAGACGGGGGGAGGCGGTGGTCGGTCGCCTCC60    CTCCTGCCCCCGGCCTGGCTTCGCGGTGGAGGCGGTGCCTCTCCGGCAAGGCAGACCAGG120    CTGGGCGGACGCGCGGCGCGGGGCGGGCTAGGGAAGGCCGGGGGCCTCGCGCTCGGGCCC180    CGGGCGGCGACTGACAGCGGCGGCGGCGGCGGCAGCGGCTCCAAGGCGAGCGTGGTCCCC240    GCGTGCGCACAAGCTCGCCGCCGCGCAGGGACCCACGGACACCGGCGCCGGGCGGACACA300    CAGACGCGGAGATCGGGCTCTACGCGCGCTACTCAGCGCACGAGCTCCCCATCCCTGGGC360    GGAGCGGGGCGCGGACTCGCCGCTGCGCGCCCTCCCCGCGGAGTCTGCCCCGGGCAGACC420    CGCAGCCCGCGGCCCCGCCGCGAGGCCCCTGGGTGAGCAGCCTGTAGACACCTGGGGTTG480    AGCAGTGGCGGCTGTGAATGAGAGGCGGGCGGCACTGGCCCGAGCCGCCTTGCAGGCTGA540    GAAGCGTCATGGCCAGCATCGCGCAGGTCTCCCTGGCTGCTCTCCTCCTGCTGCCTATGG600    CCACCGCCATGCATTCCGACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGA660    TCCAGAGGGTGAATGACCTGATGGGCTTGAATGACTCCTCCCCAGGGTGCCCTGGGATGT720    GGGACAACATCACGTGTTGGAAGCCCGCCCACGTGGGTGAGATGGTCCTGGTCAGTTGCC780    CTGAACTCTTCCGAATCTTCAACCCAGACCAAGTCTGGGAGACGGAAACCATCGGAGAGT840    TCGGTTTTGCAGACAGTAAATCCTTGGATCTCTCAGACATGAGGGTGGTGAGCCGGAATT900    GCACGGAGGATGGATGGTCAGAGCCATTCCCTCATTATTTCGATGCCTGTGGGTTTGAGG960    AGTACGAATCTGAGACTGGGGACCAGGATTACTACTACCTGTCAGTGAAGGCCCTGTACA1020    CAGTTGGCTACAGCACGTCCCTCGTCACCCTCACCACTGCCATGGTCATCCTGTGTCGTT1080    TCCGGAAGCTGCACTGCACCCGCAACTTCATCCACATGAACCTCTTCGTGTCGTTTATGC1140    TGAGGGCCATCTCCGTCTTCATCAAAGACTGGATCCTCTATGCTGAGCAGGACAGCAATC1200    ACTGCTTTGTCTCCACTGTGGAATGCAAGGCTGTGATGGTTTTCTTCCACTACTGTGTTG1260    TATCCAACTACTTCTGGCTGTTCATCGAGGGCCTGTATCTCTTCACCCTGCTGGTGGAGA1320    CCTTCTTCCCCGAGAGGAGATATTTCTACTGGTACATCATCATTGGCTGGGGGACACCAA1380    CTGTGTGTGTGTCTGTGTGGGCTATGCTGAGGCTCTACTTCGATGACACAGGCTGCTGGG1440    ATATGAATGACAACACGGCTCTGTGGTGGGTGATCAAAGGCCCTGTAGTTGGCTCCATAA1500    TGGTTAATTTTGTGCTCTTCATCGGCATCATTGTCATCCTTGTGCAGAAACTTCAGTCTC1560    CAGACATGGGAGGCAACGAGTCCAGCATCTACTTACGGCTCGCCAGGTCCACCTTGCTGC1620    TCATCCCACTCTTTGGAATCCACTACACTGTCTTTGCTTTCTCCCCGGAGAACGTCAGCA1680    AGAGGGAGAGACTGGTGTTTGAGCTGGGTCTGGGCTCCTTCCAGGGCTTTGTGGTGGCTG1740    TTCTCTATTGCTTTCTGAATGGAGAGGTGCAGGCGGAGATCAAGAGGAAGTGGCGGAGCT1800    GGAAGGTGAACCGCTACTTCACCATGGACTTCAAGCACCGGCACCCATCCCTGGCCAGCA1860    GCGGGGTGAACGGGGGCACCCAGCTCTCCATCCTGAGCAAGAGCAGCTCCCAGATCCGCA1920    TGTCTGGGCTTCCGGCCGACAACCTGGCCACCTGAGCCCACCCTGCCCCCTCCTCTCCTC1980    TGTACGCAGGCTGGGGCTGTGGTGGGGCGCCGGCCCACGCATGTTGTGCCTCTTCTCGCC2040    TTCGGGCAGGCCCCGGGCTGGGCGCCTGGCCCCCGAGGTTGGAGAAGGATGCGGGACAGG2100    CAGCTGTTTAGCCTTCCTGTTTTGGCGCTGGCCCAACCACCGTGGGTCCCTGGGCCTGCA2160    CCCAGACATGTAATACTCCTTAATTGGGAAGTCATCCATTCTTTCCCTTTCCCAAGTCCT2220    TGCTTATTAAGAGGTTCAAGTCACCTACCCAATTCAGAAGCTTAAGTAACCACTAACCAC2280    CGTGACTGCGTGGGAGGCCTCCCATGGGCTGAGCTACTGACTTGGCTTTGGGGGCCTTGG2340    GCTGGGGCCCTCCTTAAAGCCCCCCCTGAAATTGTCGGACCTCAAAGTGTGACTCCTTTG2400    AGTCTACTCGCCACCCCCGTGGCCCTTTGCAGCCCTGGTCCAGTCACCGAGGTTACTGGA2460    AGTCCAGCTTGGATGGCCAGACAGCTTTTTGGCACAGGCAGACCCATGCTCACCCAACAT2520    TTTAGTGTCCAGGTGCCCAGGTGCCCAGGTGCCCAGCTCCTGGGCATCAGACAGTGGGAA2580    AGCTCCAGGGATCTACCATTCAGAGACTTCAGTTTGGATGTAGGGCTAAGGCCAGAGAAA2640    AGTTCTGGAGCTTTTCATTTGGCCCAAGAAAAAACTGCCAAGATCCAGAAAAGTGGATCT2700    GAGTGGAATTTAGATGCAAAGAGCTTGGAG2730    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1869 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 77..1477    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    CGAGTGGACAGTGGCAGGCGGTGACTGAATCTCCAAGTCTGGAAACAATAGCCAGAGATA60    GTGGCTGGGAAGCACCATGGCCAGAGTCCTGCAGCTCTCCCTGACTGCTCTCCTGCTGCC120    TGTGGCTATTGCTATGCACTCTGACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGA180    GAGGATCCAGAGGGCCAACGACCTGATGGGACTAAACGAGTCTTCCCCAGGTTGCCCTGG240    CATGTGGGACAATATCACATGTTGGAAGCCAGCTCAAGTAGGTGAGATGGTCCTTGTAAG300    CTGCCCTGAGGTCTTCCGGATCTTCAACCCGGACCAAGTCTGGATGACAGAAACCATAGG360    AGATTCTGGTTTTGCCGATAGTAATTCCTTGGAGATCACAGACATGGGGGTCGTGGGCCG420    GAACTGCACAGAGGACGGCTGGTCGGAGCCCTTCCCCCACTACTTCGATGCTTGTGGGTT480    TGATGATTATGAGCCTGAGTCTGGAGATCAGGATTATTACTACCTGTCGGTGAAGGCTCT540    CTACACAGTCGGCTACAGCACTTCCCTCGCCACCCTCACTACTGCCATGGTCATCTTGTG600    CCGCTTCCGGAAGCTGCATTGCACTCGCAACTTCATCCACATGAACCTGTTTGTATCCTT660    CATGCTGAGGGCTATCTCCGTCTTCATCAAGGACTGGATCTTGTACGCCGAGCAGGACAG720    CAGTCACTGCTTCGTTTCCACCGTGGAGTGCAAAGCTGTCATGGTTTTCTTCCACTACTG780    CGTGGTGTCCAACTACTTCTGGCTGTTCATTGAAGGCCTGTACCTCTTTACACTGCTGGT840    GGAGACCTTCTTCCCTGAGAGGAGATATTTCTACTGGTACACCATCATCGGCTGGGGGAC900    ACCTACTGTGTGTGTAACAGTGTGGGCTGTGCTGAGGCTCTATTTTGATGATGCAGGATG960    CTGGGATATGAATGACAGCACAGCTCTGTGGTGGGTGATCAAAGGCCCCGTGGTTGGCTC1020    TATAATGGTTAACTTTGTGCTTTTCATCGGCATCATCATCATCCTTGTACAGAAGCTGCA1080    GTCCCCAGACATGGGAGGCAACGAGTCCAGCATCTACTTACGGCTGGCCCGCTCCACCCT1140    ACTGCTCATCCCACTCTTCGGAATCCACTACACAGTATTCGCCTTCTCTCCAGAGAACGT1200    CAGCAAGAGGGAAAGACTTGTGTTTGAGCTTGGGCTGGGCTCCTTCCAGGGCTTTGTGGT1260    GGCTGTACTCTACTGCTTCCTGAATGGGGAGGTACAGGCAGAGATTAAGAGGAAATGGAG1320    GAGCTGGAAGGTGAACCGTTACTTCACTATGGACTTCAAGCACCGGCACCCGTCCCTGGC1380    CAGCAGTGGAGTAAATGGGGGAACCCAGCTGTCCATCCTGAGCAAGAGCAGCTCCCAGCT1440    CCGCATGTCCAGCCTCCCGGCCGACAACTTGGCCACCTGAGGCCTGTCTCCCTCCTCCTT1500    CTGCACAGGCTGGGGCTGCGGGCCAGTGCCTGAGCATGTTTGTGCCTCTCCCCTCTCCTT1560    GGGCAGGCCCTGGGTAGGAAGCTGGGCTCCTCCCCAAAGGGGAAGAGAGAGATAGGGTAT1620    AGGCTGATATTGCTCCTCCTGTTTGGGTCCCACCTACTGTGATTCATTGAGCCTGATTTG1680    ACATGTAAATACACCTCAAATTTGGAAAGTTGCCCCATCTCTGCCCCCAACCCATGCCCC1740    TGCTCACCTCTGCCAGGCCCCAGCTCAACCTACTGTGTCAAGGCCAGCCTCAGTGATAGT1800    CTGATCCCAGGTACAAGGCCTTGTGAGCTGAGGCTGAAAGGCCTGTTTTGGAGAGGCTGG1860    GGTAGTGCC1869    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2548 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 77..1561    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    CGAGTGGACAGTGGCAGGCGGTGACTGAATCTCCAAGTCTGGAAACAATAGCCAGAGATA60    GTGGCTGGGAAGCACCATGGCCAGAGTCCTGCAGCTCTCCCTGACTGCTCTCCTGCTGCC120    TGTGGCTATTGCTATGCACTCTGACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGA180    GAGGATCCAGAGGGCCAACGACCTGATGGGACTAAACGAGTCTTCCCCAGGTTGCCCTGG240    CATGTGGGACAATATCACATGTTGGAAGCCAGCTCAAGTAGGTGAGATGGTCCTTGTAAG300    CTGCCCTGAGGTCTTCCGGATCTTCAACCCGGACCAAGTCTGGATGACAGAAACCATAGG360    AGATTCTGGTTTTGCCGATAGTAATTCCTTGGAGATCACAGACATGGGGGTCGTGGGCCG420    GAACTGCACAGAGGACGGCTGGTCGGAGCCCTTCCCCCACTACTTCGATGCTTGTGGGTT480    TGATGATTATGAGCCTGAGTCTGGAGATCAGGATTATTACTACCTGTCGGTGAAGGCTCT540    CTACACAGTCGGCTACAGCACTTCCCTCGCCACCCTCACTACTGCCATGGTCATCTTGTG600    CCGCTTCCGGAAGCTGCATTGCACTCGCAACTTCATCCACATGAACCTGTTTGTATCCTT660    CATGCTGAGGGCTATCTCCGTCTTCATCAAGGACTGGATCTTGTACGCCGAGCAGGACAG720    CAGTCACTGCTTCGTTTCCACCGTGGAGTGCAAAGCTGTCATGGTTTTCTTCCACTACTG780    CGTGGTGTCCAACTACTTCTGGCTGTTCATTGAAGGCCTGTACCTCTTTACACTGCTGGT840    GGAGACCTTCTTCCCTGAGAGGAGATATTTCTACTGGTACACCATCATCGGCTGGGGGAC900    ACCTACTGTGTGTGTAACAGTGTGGGCTGTGCTGAGGCTCTATTTTGATGATGCAGGATG960    CTGGGATATGAATGACAGCACAGCTCTGTGGTGGGTGATCAAAGGCCCCGTGGTTGGCTC1020    TATAATGGTTAACTTTGTGCTTTTCATCGGCATCATCATCATCCTTGTACAGAAGCTGCA1080    GTCCCCAGACATGGGAGGCAACGAGTCCAGCATCTACTTCAGCTGCGTGCAGAAATGCTA1140    CTGCAAGCCACAGCGGGCTCAGCAGCACTCTTGCAAGATGTCAGAACTATCCACCATTAC1200    TCTACGGCTGGCCCGCTCCACCCTACTGCTCATCCCACTCTTCGGAATCCACTACACAGT1260    ATTCGCCTTCTCTCCAGAGAACGTCAGCAAGAGGGAAAGACTTGTGTTTGAGCTTGGGCT1320    GGGCTCCTTCCAGGGCTTTGTGGTGGCTGTACTCTACTGCTTCCTGAATGGGGAGGTACA1380    GGCAGAGATTAAGAGGAAATGGAGGAGCTGGAAGGTGAACCGTTACTTCACTATGGACTT1440    CAAGCACCGGCACCCGTCCCTGGCCAGCAGTGGAGTAAATGGGGGAACCCAGCTGTCCAT1500    CCTGAGCAAGAGCAGCTCCCAGCTCCGCATGTCCAGCCTCCCGGCCGACAACTTGGCCAC1560    CTGAGGCCTGTCTCCCTCCTCCTTCTGCACAGGCTGGGGCTGCGGGCCAGTGCCTGAGCA1620    TGTTTGTGCCTCTCCCCTCTCCTTGGGCAGGCCCTGGGTAGGAAGCTGGGCTCCTCCCCA1680    AAGGGGAAGAGAGAGATAGGGTATAGGCTGATATTGCTCCTCCTGTTTGGGTCCCACCTA1740    CTGTGATTCATTGAGCCTGATTTGACATGTAAATACACCTCAAATTTGGAAAGTTGCCCC1800    ATCTCTGCCCCCAACCCATGCCCCTGCTCACCTCTGCCAGGCCCCAGCTCAACCTACTGT1860    GTCAAGGCCAGCCTCAGTGATAGTCTGATCCCAGGTACAAGGCCTTGTGAGCTGAGGCTG1920    AAAGGCCTGTTTTGGAGAGGCTGGGGTAGTGCCCACCCCAGCAGCCTTTCAGCAAATTGA1980    CTTTGGATGTGGACCCTTCTCAGCCTGTACCAAGTACTGCAGTTGGCTAGGGATGCAGCT2040    CAGTTTCCTGAGCATCCTTTGGAGCAGGTCAACCTGAGGCTCCTTTTGCTTACCCGACAT2100    CTAAGTTGTCCAGGTGCTCGGCTCCTGTGTGCCTGGATGACGGGAGGGCTCCGGGGTCTT2160    TCAGTCAAAGACTTACATTGAGGTGGGGTGAGAGTCAGAGAAAAGTTCTGGTGCTTTTCA2220    TTTGTTCTAAGAGCTGAGAGCCAGGAATGCAGAGTCAATTGGGAAGGAGATGGGATAGCT2280    GATGATCTTACCATGTCCATGACTGTGCCCCTGATTCAAGACCGGATCATGTGGTGGCTT2340    TATTTCTACACTTCTTGTCCACAATGGACAGTCTGAGGAAGCTCTTCTTTCAGCCACAAC2400    AACCACAGAAAGCCCTTTCTTCTCCCCTCTTGTTTCTCCATAAGTCAAAGCCATGTTTAG2460    AACGGACCAGCCACCTTGCGATGAAATCACTGAGTTCTGAAGCAACTTTCAATTTCCACG2520    AGCCAAGTCCTGGGTCCAGGGACGCCCC2548    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1664 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 74..1648    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    AGCCCAGAGACACATTGGGGCTGACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTG60    GCCAAGAAGTGTCATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCACTGCGGGGCCTG120    TCCGTGGGGCCGGGGCAGACTCCGCAAAGGACGCGCAGCCTGCAAGTCCGCGGCCCAGAG180    ACACATTGGGGCTGACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTGGCCAAGAAG240    TGTCATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCTCCTCCTGCTGCCTATGGCCCC300    TGCCATGCATTCTGACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGATCCA360    GAGGGCCAATGAGCTGATGGGCTTCAATGATTCCTCTCCAGGCTGTCCTGGGATGTGGGA420    CAACATCACGTGTTGGAAGCCCGCCCATGTGGGTGAGATGGTCCTGGTCAGCTGCCCTGA480    GCTCTTCCGAATCTTCAACCCAGACCAAGTCTGGGAGACCGAAACCATTGGAGAGTCTGA540    TTTTGGTGACAGTAACTCCTTAGATCTCTCAGACATGGGAGTGGTGAGCCGGAACTGCAC600    GGAGGATGGCTGGTCGGAACCCTTCCCTCATTACTTTGATGCCTGTGGGTTTGATGAATA660    TGAATCTGAGACTGGGGACCAGGATTATTACTACCTGTCAGTGAAGGCCCTCTACACGGT720    TGGCTACAGCACATCCCTCGTCACCCTCACCACTGCCATGGTCATCCTTTGTCGCTTCCG780    GAAGCTGCACTGCACACGCAACTTCATCCACATGAACCTGTTTGTGTCGTTCATGCTGAG840    GGCGATCTCCGTCTTCATCAAAGACTGGATTCTGTATGCGGAGCAGGACAGCAACCACTG900    CTTCATCTCCACTGTGGAATGTAAGGCCGTCATGGTTTTCTTCCACTACTGTGTTGTGTC960    CAACTACTTCTGGCTGTTCATCGAGGGCCTGTACCTCTTCACTCTGCTGGTGGAGACCTT1020    CTTCCCTGAAAGGAGATACTTCTACTGGTACACCATCATTGGCTGGGGGTCCCCAACTGT1080    GTGTGTGACAGTGTGGGCTACGCTGAGACTCTACTTTGATGACACAGGCTGCTGGGATAT1140    GAATGACAGCACAGCTCTGTGGTGGGTGATCAAAGGCCCTGTGGTTGGCTCTATCATGGT1200    TAACTTTGTGCTTTTTATTGGCATTATCGTCATCCTTGTGCAGAAACTTCAGTCTCCAGA1260    CATGGGAGGCAATGAGTCCAGCATCTACTTGCGACTGGCCCGGTCCACCCTGCTGCTCAT1320    CCCACTATTCGGAATCCACTACACAGTATTTGCCTTCTCCCCAGAGAATGTCAGCAAAAG1380    GGAAAGACTCGTGTTTGAGCTGGGGCTGGGCTCCTTCCAGGGCTTTGTGGTGGCTGTTCT1440    CTACTGTTTTCTGAATGGTGAGGTACAAGCGGAGATCAAGCGAAAATGGCGAAGCTGGAA1500    GGTGAACCGTTACTTCGCTGTGGACTTCAAGCACCGACACCCGTCTCTGGCCAGCAGTGG1560    GGTGAATGGGGGCACCCAGCTCTCCATCCTGAGCAAGAGCAGCTCCCAAATCCGCATGTC1620    TGGCCTCCCTGCTGACAATCTGGCCACCTGAGCCATGCTCCCCT1664    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1748 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 74..1732    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    AGCCCAGAGACACATTGGGGCTGACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTG60    GCCAAGAAGTGTCATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCACTGCGGGGCCTG120    TCCGTGGGGCCGGGGCAGACTCCGCAAAGGACGCGCAGCCTGCAAGTCCGCGGCCCAGAG180    ACACATTGGGGCTGACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTGGCCAAGAAG240    TGTCATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCTCCTCCTGCTGCCTATGGCCCC300    TGCCATGCATTCTGACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGATCCA360    GAGGGCCAATGAGCTGATGGGCTTCAATGATTCCTCTCCAGGCTGTCCTGGGATGTGGGA420    CAACATCACGTGTTGGAAGCCCGCCCATGTGGGTGAGATGGTCCTGGTCAGCTGCCCTGA480    GCTCTTCCGAATCTTCAACCCAGACCAAGTCTGGGAGACCGAAACCATTGGAGAGTCTGA540    TTTTGGTGACAGTAACTCCTTAGATCTCTCAGACATGGGAGTGGTGAGCCGGAACTGCAC600    GGAGGATGGCTGGTCGGAACCCTTCCCTCATTACTTTGATGCCTGTGGGTTTGATGAATA660    TGAATCTGAGACTGGGGACCAGGATTATTACTACCTGTCAGTGAAGGCCCTCTACACGGT720    TGGCTACAGCACATCCCTCGTCACCCTCACCACTGCCATGGTCATCCTTTGTCGCTTCCG780    GAAGCTGCACTGCACACGCAACTTCATCCACATGAACCTGTTTGTGTCGTTCATGCTGAG840    GGCGATCTCCGTCTTCATCAAAGACTGGATTCTGTATGCGGAGCAGGACAGCAACCACTG900    CTTCATCTCCACTGTGGAATGTAAGGCCGTCATGGTTTTCTTCCACTACTGTGTTGTGTC960    CAACTACTTCTGGCTGTTCATCGAGGGCCTGTACCTCTTCACTCTGCTGGTGGAGACCTT1020    CTTCCCTGAAAGGAGATACTTCTACTGGTACACCATCATTGGCTGGGGGACCCCAACTGT1080    GTGTGTGACAGTGTGGGCTACGCTGAGACTCTACTTTGATGACACAGGCTGCTGGGATAT1140    GAATGACAGCACAGCTCTGTGGTGGGTGATCAAAGGCCCTGTGGTTGGCTCTATCATGGT1200    TAACTTTGTGCTTTTTATTGGCATTATCGTCATCCTTGTGCAGAAACTTCAGTCTCCAGA1260    CATGGGAGGCAATGAGTCCAGCATCTACTTCAGCTGCGTGCAGAAATGCTACTGCAAGCC1320    ACAGCGGGCTCAGCAGCACTCTTGCAAGATGTCAGAACTGTCCACCATTACTCTGCGACT1380    GGCCCGGTCCACCCTGCTGCTCATCCCACTATTCGGAATCCACTACACAGTATTTGCCTT1440    CTCCCCAGAGAATGTCAGCAAAAGGGAAAGACTCGTGTTTGAGCTGGGGCTGGGCTCCTT1500    CCAGGGCTTTGTGGTGGCTGTTCTCTACTGTTTTCTGAATGGTGAGGTACAAGCGGAGAT1560    CAAGCGAAAATGGCGAAGCTGGAAGGTGAACCGTTACTTCGCTGTGGACTTCAAGCACCG1620    ACACCCGTCTCTGGCCAGCAGTGGGGTGAATGGGGGCACCCAGCTCTCCATCCTGAGCAA1680    GAGCAGCTCCCAAATCCGCATGTCTGGCCTCCCTGCTGACAATCTGGCCACCTGAGCCAT1740    GCTCCCCT1748    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1745 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 74..1729    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    AGCCCAGAGACACATTGGGGCTGACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTG60    GCCAAGAAGTGTCATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCACTGCGGGGCCTG120    TCCGTGGGGCCGGGGCAGACTCCGCAAAGGACGCGCAGCCTGCAAGTCCGCGGCCCAGAG180    ACACATTGGGGCTGACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTGGCCAAGAAG240    TGTCATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCTCCTCCTGCTGCCTATGGCCCC300    TGCCATGCATTCTGACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGATCCA360    GAGGGCCAATGAGCTGATGGGCTTCAATGATTCCTCTCCAGGCTGTCCTGGGATGTGGGA420    CAACATCACGTGTTGGAAGCCCGCCCATGTGGGTGAGATGGTCCTGGTCAGCTGCCCTGA480    GCTCTTCCGAATCTTCAACCCAGACCAAGTCTGGGAGACCGAAACCATTGGAGAGTCTGA540    TTTTGGTGACAGTAACTCCTTAGATCTCTCAGACATGGGAGTGGTGAGCCGGAACTGCAC600    GGAGGATGGCTGGTCGGAACCCTTCCCTCATTACTTTGATGCCTGTGGGTTTGATGAATA660    TGAATCTGAGACTGGGGACCAGGATTATTACTACCTGTCAGTGAAGGCCCTCTACACGGT720    TGGCTACAGCACATCCCTCGTCACCCTCACCACTGCCATGGTCATCCTTTGTCGCTTCCG780    GAAGCTGCACTGCACACGCAACTTCATCCACATGAACCTGTTTGTGTCGTTCATGCTGAG840    GGCGATCTCCGTCTTCATCAAAGACTGGATTCTGTATGCGGAGCAGGACAGCAACCACTG900    CTTCATCTCCACTGTGGAATGTAAGGCCGTCATGGTTTTCTTCCACTACTGTGTTGTGTC960    CAACTACTTCTGGCTGTTCATCGAGGGCCTGTACCTCTTCACTCTGCTGGTGGAGACCTT1020    CTTCCCTGAAAGGAGATACTTCTACTGGTACACCATCATTGGCTGGGGGACCCCAACTGT1080    GTGTGTGACAGTGTGGGCTACGCTGAGACTCTACTTTGATGACACAGGCTGCTGGGATAT1140    GAATGACAGCACAGCTCTGTGGTGGGTGATCAAAGGCCCTGTGGTTGGCTCTATCATGGT1200    TAACTTTGTGCTTTTTATTGGCATTATCGTCATCCTTGTGCAGAAACTTCAGTCTCCAGA1260    CATGGGAGGCAATGAGTCCAGCATCTACTTCTGCGTGCAGAAATGCTACTGCAAGCCACA1320    GCGGGCTCAGCAGCACTCTTGCAAGATGTCAGAACTGTCCACCATTACTCTGCGACTGGC1380    CCGGTCCACCCTGCTGCTCATCCCACTATTCGGAATCCACTACACAGTATTTGCCTTCTC1440    CCCAGAGAATGTCAGCAAAAGGGAAAGACTCGTGTTTGAGCTGGGGCTGGGCTCCTTCCA1500    GGGCTTTGTGGTGGCTGTTCTCTACTGTTTTCTGAATGGTGAGGTACAAGCGGAGATCAA1560    GCGAAAATGGCGAAGCTGGAAGGTGAACCGTTACTTCGCTGTGGACTTCAAGCACCGACA1620    CCCGTCTCTGGCCAGCAGTGGGGTGAATGGGGGCACCCAGCTCTCCATCCTGAGCAAGAG1680    CAGCTCCCAAATCCGCATGTCTGGCCTCCCTGCTGACAATCTGGCCACCTGAGCCATGCT1740    CCCCT1745    (2) INFORMATION FOR SEQ ID NO:45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1748 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 74..1732    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    AGCCCAGAGACACATTGGGGCTGACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTG60    GCCAAGAAGTGTCATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCACTGCGGGGCCTG120    TCCGTGGGGCCGGGGCAGACTCCGCAAAGGACGCGCAGCCTGCAAGTCCGCGGCCCAGAG180    ACACATTGGGGCTGACCTGCCGCTGCTGTCAGTGGGAGGCCAGTGGTGCTGGCCAAGAAG240    TGTCATGGCTGGTGTCGTGCACGTTTCCCTGGCTGCTCTCCTCCTGCTGCCTATGGCCCC300    TGCCATGCATTCTGACTGCATCTTCAAGAAGGAGCAAGCCATGTGCCTGGAGAAGATCCA360    GAGGGCCAATGAGCTGATGGGCTTCAATGATTCCTCTCCAGGCTGTCCTGGGATGTGGGA420    CAACATCACGTGTTGGAAGCCCGCCCATGTGGGTGAGATGGTCCTGGTCAGCTGCCCTGA480    GCTCTTCCGAATCTTCAACCCAGACCAAGTCTGGGAGACCGAAACCATTGGAGAGTCTGA540    TTTTGGTGACAGTAACTCCTTAGATCTCTCAGACATGGGAGTGGTGAGCCGGAACTGCAC600    GGAGGATGGCTGGTCGGAACCCTTCCCTCATTACTTTGATGCCTGTGGGTTTGATGAATA660    TGAATCTGAGACTGGGGACCAGGATTATTACTACCTGTCAGTGAAGGCCCTCTACACGGT720    TGGCTACAGCACATCCCTCGTCACCCTCACCACTGCCATGGTCATCCTTTGTCGCTTCCG780    GAAGCTGCACTGCACACGCAACTTCATCCACATGAACCTGTTTGTGTCGTTCATGCTGAG840    GGCGATCTCCGTCTTCATCAAAGACTGGATTCTGTATGCGGAGCAGGACAGCAACCACTG900    CTTCATCTCCACTGTGGAATGTAAGGCCGTCATGGTTTTCTTCCACTACTGTGTTGTGTC960    CAACTACTTCTGGCTGTTCATCGAGGGCCTGTACCTCTTCACTCTGCTGGTGGAGACCTT1020    CTTCCCTGAAAGGAGATACTTCTACTGGTACACCATCATTGGCTGGGGGACCCCAACTGT1080    GTGTGTGACAGTGTGGGCTACGCTGAGACTCTACTTTGATGACACAGGCTGCTGGGATAT1140    GAATGACAGCACAGCTCTGTGGTGGGTGATCAAAGGCCCTGTGGTTGGCTCTATCATGGT1200    TAACTTTGTGCTTTTTATTGGCATTATCGTCATCCTTGTGCAGAAACTTCAGTCTCCAGA1260    CATGGGAGGCAATGAGTCCAGCATCTACTTAACAAATTTAAGCCCGCGAGTCCCCAAGAA1320    AGCCCGAGAGGACCCCCTGCCTGTGCCCTCAGACCAGCATTCACTCCCTTTCCTGCGACT1380    GGCCCGGTCCACCCTGCTGCTCATCCCACTATTCGGAATCCACTACACAGTATTTGCCTT1440    CTCCCCAGAGAATGTCAGCAAAAGGGAAAGACTCGTGTTTGAGCTGGGGCTGGGCTCCTT1500    CCAGGGCTTTGTGGTGGCTGTTCTCTACTGTTTTCTGAATGGTGAGGTACAAGCGGAGAT1560    CAAGCGAAAATGGCGAAGCTGGAAGGTGAACCGTTACTTCGCTGTGGACTTCAAGCACCG1620    ACACCCGTCTCTGGCCAGCAGTGGGGTGAATGGGGGCACCCAGCTCTCCATCCTGAGCAA1680    GAGCAGCTCCCAAATCCGCATGTCTGGCCTCCCTGCTGACAATCTGGCCACCTGAGCCAT1740    GCTCCCCT1748    (2) INFORMATION FOR SEQ ID NO:46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgLysGln    151015    MetAlaValLysLysTyrLeuAlaAlaValLeuGlyLysArgTyrLys    202530    GlnArgValLysAsnLys    35    (2) INFORMATION FOR SEQ ID NO:47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgLysGln    151015    MetAlaValLysLysTyrLeuAlaAlaValLeu    2025    (2) INFORMATION FOR SEQ ID NO:48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 37 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid, Synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    CAGAAAGCTTCGGACCATGCGCCCTCCGAGCCCACCG37    (2) INFORMATION FOR SEQ ID NO:49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 37 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid, Synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    GGGCTCTAGACGGTCAGACCAGGGAGACCTCCGCTTG37    (2) INFORMATION FOR SEQ ID NO:50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    AspCysIlePheLysLysGluGlnAlaMetCysLeuGluLysIleGln    151015    (2) INFORMATION FOR SEQ ID NO:51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid, Synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    TGGATCTTCTCCAGGTGCATDGCCTGCTCCTTCTTGAAGATGTGGTC47    (2) INFORMATION FOR SEQ ID NO:52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid, Synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:    CTGGGATATGAATGACAGCACAGC24    (2) INFORMATION FOR SEQ ID NO:53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid, Synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:    TCTGGGGAGAAGGCAAATACTGTG24    (2) INFORMATION FOR SEQ ID NO:54:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid, Synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:    TGCGTGCAGAAATGCTACTGCAAGCCACAG30    (2) INFORMATION FOR SEQ ID NO:55:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid, Synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:    GACCCCCTGCCTGTGCCCTCAGACCAGCAT30    (2) INFORMATION FOR SEQ ID NO:56:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1324 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:    GlyGlxMetArgProProSerProProHisValArgTrpLeuCysVal    151015    LeuAlaGlyAlaLeuAlaCysAlaLeuArgProAlaGlySerGlnAla    202530    AlaSerProGlnHisGluCysGluTyrLeuGlnLeuIleGluIleGln    354045    ArgGlnGlnCysLeuGluGluAlaGlnLeuGluAsnGluThrThrGly    505560    CysSerLysMetTrpAspAsnLeuThrCysTrpProThrThrProArg    65707580    GlyGlnAlaValValLeuAspCysProLeuIlePheGlnLeuPheAla    859095    ProIleHisGlyTyrAsnIleSerArgSerCysThrGluGluGlyTrp    100105110    SerGlnLeuGluProGlyProTyrHisIleAlaCysGlyLeuAsnAsp    115120125    ArgAlaSerSerLeuAspGluGlnGlnGlnThrLysPheTyrAsnThr    130135140    ValLysThrGlyTyrThrIleGlyTyrSerLeuSerLeuAlaSerLeu    145150155160    LeuValAlaMetAlaIleLeuSerLeuPheArgLysLeuHisCysThr    165170175    ArgAsnTyrIleHisMetHisLeuPheMetSerPheIleLeuArgAla    180185190    ThrAlaValPheIleLysAspMetAlaLeuPheAsnSerGlyGluIle    195200205    AspHisCysSerGluAlaSerValGlyCysLysAlaAlaValValPhe    210215220    PheGlnTyrCysValMetAlaAsnPhePheTrpLeuLeuValGluGly    225230235240    LeuTyrLeuTyrThrLeuLeuAlaValSerPhePheSerGluArgLys    245250255    TyrPheTrpGlyTyrIleLeuIleGlyTrpGlyValProSerValPhe    260265270    IleThrIleTrpThrValValArgIleTyrPheGluAspPheGlyCys    275280285    TrpAspThrIleIleAsnSerSerLeuTrpTrpIleIleLysAlaPro    290295300    IleLeuLeuSerIleLeuValAsnPheValLeuPheIleCysIleIle    305310315320    ArgIleLeuValGlnLysLeuArgProProAspIleGlyLysAsnAsp    325330335    SerSerProTyrSerArgLeuAlaLysSerThrLeuLeuLeuIlePro    340345350    LeuPheGlyIleHisTyrValMetPheAlaPhePheProAspAsnPhe    355360365    LysAlaGlnValLysMetValPheGluLeuValValGlySerPheGln    370375380    GlyPheValValAlaIleLeuTyrCysPheLeuAsnGlyGluValGln    385390395400    AlaGluLeuArgArgLysTrpArgArgTrpHisLeuGlnGlyValLeu    405410415    GlyTrpSerSerLysSerGlnHisProTrpGlyGlySerAsnGlyAla    420425430    ThrCysSerThrGlnValSerMetLeuThrArgValSerProSerAla    435440445    ArgArgSerSerSerPheGlnAlaGluValSerLeuValAsnHisAla    450455460    AspProPheAsnAsxCysTrpIleAsnAspTrpSerThrGluMetPro    465470475480    AlaArgAspAsnAsxAlaSerGluGlnGluAsnCysGluAspGlyThr    485490495    AsnArgSerAlaMetArgProProSerProProHisValArgTrpLeu    500505510    CysValLeuAlaGlyAlaLeuAlaCysAlaLeuArgProAlaGlySer    515520525    GlnAlaAlaSerProGlnHisGluCysGluTyrLeuGlnLeuIleGlu    530535540    IleGlnArgGlnGlnCysLeuGluGluAlaGlnLeuGluAsnGluThr    545550555560    ThrGlyCysSerLysMetTrpAspAsnLeuThrCysTrpProThrThr    565570575    ProArgGlyGlnAlaValValLeuAspCysProLeuIlePheGlnLeu    580585590    PheAlaProIleHisGlyTyrAsnIleSerArgSerCysThrGluGlu    595600605    GlyTrpSerGlnLeuGluProGlyProTyrHisIleAlaCysGlyLeu    610615620    AsnAspArgAlaSerSerLeuAspGluGlnGlnGlnThrLysPheTyr    625630635640    AsnThrValLysThrGlyTyrThrIleGlyTyrSerLeuSerLeuAla    645650655    SerLeuLeuValAlaMetAlaIleLeuSerLeuPheArgLysLeuHis    660665670    CysThrArgAsnTyrIleHisMetHisLeuPheMetSerPheIleLeu    675680685    ArgAlaThrAlaValPheIleLysAspMetAlaLeuPheAsnSerGly    690695700    GluIleAspHisCysSerGluAlaSerValGlyCysLysAlaAlaVal    705710715720    ValPhePheGlnTyrCysValMetAlaAsnPhePheTrpLeuLeuVal    725730735    GluGlyLeuTyrLeuTyrThrLeuLeuAlaValSerPhePheSerGlu    740745750    ArgLysAsnAsxAsnAsxAspLeuThrMetArgProProSerProPro    755760765    HisValArgTrpLeuCysValLeuAlaGlyAlaLeuAlaCysAlaLeu    770775780    ArgProAlaGlySerGlnAlaAlaSerProGlnHisGluCysGluTyr    785790795800    LeuGlnLeuIleGluIleGlnArgGlnGlnCysLeuGluGluAlaGln    805810815    LeuGluAsnGluThrThrGlyCysSerLysMetTrpAspAsnLeuThr    820825830    CysTrpProThrThrProArgGlyGlnAlaValValLeuAspCysPro    835840845    LeuIlePheGlnLeuPheAlaProIleHisGlyTyrAsnIleSerArg    850855860    SerCysThrGluGluGlyTrpSerGlnLeuGluProGlyProTyrHis    865870875880    IleAlaCysGlyLeuAsnAspArgAlaSerSerLeuAspGluGlnGln    885890895    GlnThrLysPheTyrAsnThrValLysThrGlyTyrThrIleGlyTyr    900905910    SerLeuSerLeuAlaSerLeuLeuValAlaMetAlaIleLeuSerLeu    915920925    PheArgLysLeuHisCysThrArgAsnTyrIleHisMetHisLeuPhe    930935940    MetSerPheIleLeuArgAlaThrAlaValPheIleLysAspMetAla    945950955960    LeuPheAsnSerGlyGluIleAspHisCysSerGluAlaSerValGly    965970975    CysLysAlaAlaValValPhePheGlnTyrCysValMetAlaAsnPhe    980985990    PheTrpLeuLeuValGluGlyLeuTyrLeuTyrThrLeuLeuAlaVal    99510001005    SerPhePheSerGluArgLysArgProProAsnAsxAlaAsnAsxAsn    101010151020    AsxAlaMetTrpAlaAspProMetArgProProSerProProHisVal    1025103010351040    ArgTrpLeuCysValLeuAlaGlyAlaLeuAlaCysAlaLeuArgPro    104510501055    AlaGlySerGlnAlaAlaSerProGlnHisGluCysGluTyrLeuGln    106010651070    LeuIleGluIleGlnArgGlnGlnCysLeuGluGluAlaGlnLeuGlu    107510801085    AsnGluThrThrGlyCysSerLysMetTrpAspAsnLeuThrCysTrp    109010951100    ProThrThrProArgGlyGlnAlaValValLeuAspCysProLeuIle    1105111011151120    PheGlnLeuPheAlaProIleHisGlyTyrAsnIleSerArgSerCys    112511301135    ThrGluGluGlyTrpSerGlnLeuGluProGlyProTyrHisIleAla    114011451150    CysGlyLeuAsnAspArgAlaSerSerLeuAspGluGlnGlnGlnThr    115511601165    LysPheTyrAsnThrValLysThrGlyTyrThrIleGlyTyrSerLeu    117011751180    SerLeuAlaSerLeuLeuValAlaMetAlaIleLeuSerLeuPheArg    1185119011951200    LysLeuHisCysThrArgAsnTyrIleHisMetHisLeuPheMetSer    120512101215    PheIleLeuArgAlaThrAlaValPheIleLysAspMetAlaLeuPhe    122012251230    AsnSerGlyGluIleAspHisCysSerGluAlaSerValGlyCysLys    123512401245    AlaAlaValValPhePheGlnTyrCysValMetAlaAsnPhePheTrp    125012551260    LeuLeuValGluGlyLeuTyrLeuTyrThrLeuLeuAlaValSerPhe    1265127012751280    PheSerGluArgLysThrProIleAspGlyIleAspAlaAsnPheAsp    128512901295    AlaAlaGluAspGluCysArgGluPheThrTrpAspSerIleAspSer    130013051310    IleCysPheMetTrpAspMetSerTrpAspTrpAsp    13151320    __________________________________________________________________________

What is claimed is:
 1. An isolated DNA coding for a receptor proteincapable of binding a pituitary adenylate cyclase activating protein,wherein the receptor protein comprises the amino acid sequence of SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28 or SEQ ID NO:
 29. 2. The DNAof claim 1 wherein the DNA is a cDNA.
 3. A vector containing the DNA asclaimed in claim
 1. 4. A transformed host cell containing the DNA asclaimed in claim
 1. 5. A method for preparing the receptor protein orthe salt thereof as claimed in claim 1 comprising cultivating atransformed host cell containing a DNA encoding said protein underconditions suitable for expression of said protein and recovering saidprotein.
 6. An isolated DNA comprising the nucleotide sequence of SEQ IDNO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37.